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PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT
DIAPHRAGM DURING MECHANICAL VENTILATION
R. ANDREW SHANELY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
R. Andrew Shanely
The completion of this Dissertation is one my greatest accomplishments and is dedicated
to my parents. If it were not for your support and enthusiasm of my educational
development I would never have contemplated postgraduate work, let alone completed it.
Each of you has given me the love, understanding, encouragement, and freedom to lead a
happy and successful life. Thank you.
This Dissertation is also dedicated to Shannon. Your love, support, and daily
encouragement never ceases to amaze me. My love, thank you for your day-to-day help
and understanding throughout my postgraduate career.
I would like to acknowledge those who made the successful completion of this
project possible: my mentor, Dr. Scott Powers; and my committee members, Dr. Stephen
Dodd, Dr. Randy Braith, and Dr. Paul Davenport. Collectively and individually, my
committee provided me with invaluable guidance, instruction, and patience. I would also
like to acknowledge Darin Van Gammeren, Michael McKenzie, and Murat Zergeroglu
for their contribution to this project, without which these experiments would not have
been possible. Kevin Yarasheski's measurement of [13C]leucine incorporation into
diaphragmatic proteins was the crux of these experiments. His enthusiastic participation
assured the success of these experiments. Fadia Hadad's knowledge and patience made
the measurement of myosin heavy chain mRNA a reality. Jeff Coombes, Haydar
Demirel, and Hisashi Natio must be acknowledged for their immediate guidance upon my
arrival in Dr. Scott Power's laboratory and thus their indirect contribution to this project.
I am forever indebted to Dr. Powers for serving as my mentor. His motivation,
guidance, support, mentorship, and friendship have been unflagging. A student is only
capable of what his mentor demands!
This work was made possible by funding from the National Institutes of Health
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF TABLES ............................................. .. .. .... .............. viii
LIST OF FIGURES ......... ........................................... ............ ix
A B ST R A C T .......... ..... ...................................................................................... x
1 IN TR OD U CTION .............................................. .. ........... .............. .
Objectives of Specific Aim #1 .................. ...... ........................................... 2
Aim #1: Rationale for Experimental Approach and Hypothesis ................................... 2
Objectives of Specific A im #2 .................. ................. .............. ........ .............. 3
Aim #2: Rationale for Experimental Approach and Hypothesis ................................... 3
2 LITER A TU R E R EV IEW ................................................................. ....................... 4
H history of M mechanical V entilation........................................................................... 4
Indication for Clinical Use of Mechanical Ventilation........................................... 6
M odes of M echanical Ventilator Operation ........................................ .............. 8
Controlled M echanical V entilation................................................. ................. 8
A ssist-Control V entilation ........................... ....... .................................... 9
Intermittent M mandatory Ventilation ......................... ...................................... 9
Pressure Support Ventilation ................................... ....... .............. 10
Diaphragmatic Motion during Mechanical Ventilation............................................... 10
W meaning from M echanical V entilation............................................... .... .. .............. 12
Properties of the D iaphragm .......................................................... .............. 14
Function of the D iaphragm .................................... ................................................. 14
Metabolic Characteristics of the Diaphragm ......................................................... 15
Skeletal Muscle Fiber Types within the Diaphragm............................................ 16
O x idativ e C ap city ....................................................................... .................... 18
M uscle A trophy ...................................... ............ ......................... 19
M odels of Locom otor M uscle Atrophy ................................. .................................... 20
Reduced Electrical Activation and Load Bearing........................................... 20
Reduced Loading ........................................................... 22
M models of Inactivity .................. ............................ .. ...... .. .......... .. 24
M odels of Atrophy in the Diaphragm .................................... ................... ...... ...... 26
Procedure for Investigating Diaphragmatic Atrophy........................................... 27
Electrom yographic A activity ...................... .... ............ .................. .............. 27
Diaphragmatic Mass.......................... .. ... .. .................... 28
Differential Fiber Type Response to Diaphragmatic Inactivity.......................... 28
M yosin H eavy C hain C ontent...................................................... .... .. .............. 29
M yosin H eavy C hain m R N A ........................................................................ ... 29
C ontractile P roperties....................................................... ............. .......... 30
O xidative Capacity .................. ..................................... .. .......... .. 30
Protein Synthesis......................................... .......... 30
D iaphragm L ength C hanges................... .......................... .............. ... 31
Mechanical Ventilation and Diaphragmatic Atrophy ............................................. 32
E electrical A activity ............ .......... .................. ............ ... ............. .... ............. 32
D iaphragm atic D isuse A trophy..................................... ......................... .. ....... 32
Anim al M odels and M echanical Ventilation .................. .............................. ... 33
Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties........ 34
Mechanical Ventilation and Diaphragmatic Atrophy ........................................ 35
Sum m ary of L literature R eview .......................................................................... ..... 37
3 M E T H O D S ........................................................... ................ 4 0
Experim ental D esign-Specific Aim #1 ........................................ ....... .............. 40
A nim als and Experim ental D esign............................................... ... ... .............. 40
M mechanical ventilation protocol ........................................... ............... 41
Postm ortem exam nation ................................................. ............... .... 43
Control animals (nonmechanically ventilated) protocol..............................43
M methods U sed: B iochem ical A ssays................................................ ... ................. 44
Tissue rem oval and storage............................................ ............... ... 44
Rates of in vivo diaphragmatic protein synthesis.....................................44
Statistical analy sis .................................................. ............ ...................... 49
Experim ental D esign-Specific A im #2 ................................... ................................... 49
M methods U sed: B iochem ical A ssays................................................ ... ................. 49
T otal R N A isolation ........... .............................................. ........ .............. 49
R everse transcription (R T)........................................ ........................... 50
Polymerase chain reaction (PCR) ................................................ 51
A analysis of gels ............. ......... ................ ...............................................52
Statistical analysis ....... ................................ .. .. .... ...... .. ........ .... 53
4 R E S U L T S ................................................................................................................. 5 4
Morphological, Physiological, and Post Mortem Observations ................................. 54
Influence of Mechanical Ventilation on Protein Synthesis......................................... 55
Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous
Breathing and M mechanical Ventilation .......................................... ..... ......... 57
5 D IS C U S S IO N ........................................................................................................... 7 1
O verview of Principle Findings ............................................................... ............... ... 71
Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm .............. 71
M ixed M uscle Protein Synthesis.................................................. .................... 71
M yosin H eavy Chain Protein Synthesis............................................. ... ................. 72
M yosin H eavy C hain m R N A ........................................................................ ... 74
R regulation of P rotein Synthesis.......................................................... .... ................ 75
Transcription ..... ................................ ................... 75
Translation Initiation.................................. .......................... ......76
Translation Elongation and Termination ............................... ................ 78
Critique of the Experim ental M odel ..................................... ........................ .......... 79
Nutritional Status ......................................... .............. 80
A anesthesia ..................................................................................................... 80
Infusion period ............................................... ............. .......... 81
Blood Removal ......................... ......... .............. 82
M ode of M echanical V entilation ........................................ ....................... 83
Sum m ary and Future Experim ents...................................................... .... .. .............. 83
L IST O F R E FE R E N C E S ....................................................................... ... ...................85
BIOGRAPHICAL SKETCH ............................................................. ............... 103
LIST OF TABLES
2-1 Fiber type composition (%) of the diaphragm and locomotor skeletal muscles. ........17
2-2 Bioenergetic enzyme activities in the costal diaphragm and two locomotor
m u sc le s ...................................................................... 1 9
3-1 Oligonucleotide primers used for the PCR reactions ...............................................53
4-1 Animal body mass before and after experimental period.........................................58
4-2 Heart rate response during M V and SB.................................................................... 59
4-3 Systolic blood pressure response during MV and SB ............................................59
4-4 Fractional synthetic rates of mixed muscle protein and myosin heavy chain
protein by calculation with each surrogate of the [13C]leucyl-tRNA precursor
p o o l. ........................................ .................... ................ 6 5
4-5 Total RNA obtained from the costal diaphragm ............................... ............... .66
LIST OF FIGURES
3-1 Experim mental design for Specific Aim #1. ........................................ .....................41
3-2 Schematic representation of the myosin heavy chain (MHC) genes...........................53
4-1 Plasma [13C]leucine and plasma [13C]ketoisocaproic acid ([13C]KIC) enrichment. ...60
4-2 Tissue fluid [13C]leucine enrichment in the diaphragm ............... .................. 61
4-3 Mixed muscle protein and myosin heavy chain [13C]leucine enrichment in the
diaphragm ....................................................... ................. 62
4-4 Fractional synthetic rates of mixed muscle protein (MMP) by calculation with
tissue fluid [13C leucinee. ............................................................. .....................63
4-5 Fractional synthetic rates of myosin heavy chain (MHC) protein by calculation
w ith tissue fluid [13C leucinee. ........................................ .......................... 64
4-6 R T-PCR products. ......................... ........................ .. ............. ......... 67
4-7 Relative type I M HC expression. ........................................ ........................... 67
4-8 Relative type IIa M H C expression. ........................................ ......................... 68
4-9 Relative type IIx M HC expression. ........................................................................... 69
4-10 Relative type IIb M HC expression. ........................................ ....................... 70
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT
DIAPHRAGM DURING MECHANICAL VENTILATION
R. Andrew Shanely
Chair: Scotty K. Powers, Ph.D., Ed.D.
Department: Exercise and Sport Sciences
The purpose of these experiments was to test the hypothesis that mechanical
ventilation-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of
total protein synthesis and myosin heavy chain (MHC) protein synthesis. We also tested
the hypothesis that mechanical ventilation (MV) alters pretranslational events in the
diaphragm. To test these hypotheses, we randomly assigned specific-pathogen-free
barrier-protected 4-month-old female Sprague-Dawley rats to one of three experimental
groups: MV; spontaneously breathing (SB); or control/acute anesthesia. The MV animals
were mechanically ventilated for 6, 12, or 18 hours (n=10 for each time period).
Spontaneously breathing animals underwent the same surgical procedures and were
anesthetized for the same period of time as the animals in the MV group, but were not
exposed to MV. The acute-control animals (n=10) were not exposed to MV or prolonged
anesthesia. The rate of protein synthesis was determined by measuring the rate of
[13C]leucine incorporation into total protein and MHC protein in the diaphragm of the
MV and SB rats. We isolated total RNA from the diaphragms of all groups and measured
the expression of type I, IIa, IIx, and IIb (the four adult MHC mRNA isoforms). The rate
of protein synthesis of the MV rats was compared to that of the SB rats at the 6, 12, and
18 hour time points. Six hours of MV caused a significant decrease (30%, p < 0.05) in the
rate of total protein synthesis and a significant decrease (65%, p < 0.05) in the rate of
MHC protein synthesis. The decrease (p < 0.05) in protein synthesis remained at this
depressed level after 12 and 18 hours of MV. Expression of MHC mRNA isoforms in the
diaphragms of MV animals and the SB animals did not change (p > 0.05). These data
support the hypothesis that a decrease in protein synthesis contributes to MV-induced
diaphragmatic atrophy. In contrast, these data do not support the hypothesis that MV
alters pretranslational events in the diaphragm.
Mechanical ventilation (MV) provides a means of supporting blood gas
homeostasis for patients who cannot maintain adequate alveolar ventilation.
Unfortunately, prolonged MV (i.e., > 3 days) is not without consequence because as
many as 20% of patients experience difficulty in "weaning" from the ventilator (94).
While the underlying cause for weaning difficulties has yet to be fully elucidated,
respiratory muscle atrophy and the associated contractile dysfunction are potential
In this regard, Anzueto et al. (8) reported significant reductions in diaphragmatic
strength and endurance of healthy baboons after 11 days of MV. Despite these important
physiological findings, Anzueto and colleagues' report did not investigate biochemical or
histological alterations in the diaphragm associated with MV. Further, Le Bourdelles et
al. (92) examined the effects of 48 hours of controlled MV on both atrophy and
contractile properties in the rat diaphragm. They reported a significant reduction in
isometric force generation and a reduction in both diaphragmatic mass (i.e., atrophy) and
protein content (92). Powers et al. (125) have also reported that MV leads to progressive
diaphragmatic contractile dysfunction. These experiments demonstrated a significant
correlation between time on the ventilator and contractile dysfunction (i.e., the greater the
time on the ventilator the greater the degree of diaphragmatic contractile dysfunction)
(125). Finally, recent experiments in our laboratory have demonstrated that MV for little
as 18 hours results in diaphragmatic atrophy (145). Preliminary experiments in our
laboratory suggest that the observed diaphragmatic atrophy is associated with a decreased
rate of diaphragmatic protein synthesis and a decrease in myosin heavy chain (MHC)
content. These observations form the basis for the proposed experiments.
Objectives of Specific Aim #1
The effect of MV on diaphragmatic protein synthesis was determined. We tested
the hypothesis that MV-induced diaphragmatic atrophy is due, at least in part, to a
decreased rate of total and myofibrillar protein synthesis.
Aim #1: Rationale for Experimental Approach and Hypothesis
Preliminary experiments in our laboratory indicated that prolonged MV results in
significant diaphragmatic atrophy. The extent to which decreases in protein synthesis
contribute to the MV-induced loss of diaphragmatic contractile protein is unknown.
Therefore, these experiments were designed to determine the time course of changes in
protein synthesis during 6, 12, and 18 hours of MV. This was achieved by measuring
both total and contractile protein synthesis rates (in vivo) in the diaphragms of control and
MV animals. Specifically, diaphragmatic protein synthesis was measured over the course
of the last 6 hours of the experimental period (e.g., 12 tol8 hours of MV). The fractional
rate of diaphragm muscle protein synthesis was measured using intravenous infusion of
[1-13C]leucine. We quantified the in vivo rate of incorporation of [1-13C]leucine into both
total and contractile proteins in the diaphragm by gas chromatography-combustion-
isotope ratio mass spectrometry (GC-C-IRMS). The stable isotope [1-13C]leucine was
chosen for several reasons:
* Less isotope effect (i.e., less tissue injury).
* Small tissue sample is required for analysis.
* Validity and reliability of this label for measurement of the rate of in vivo muscle
protein synthesis is well established (121).
Objectives of Specific Aim #2
The effect of MV on diaphragmatic myosin heavy chain (MHC) mRNA content
was determined. We tested the hypothesis that MV alters pretranslational events in the
Aim #2: Rationale for Experimental Approach and Hypothesis
Preliminary experiments suggested that prolonged MV results in a decrease in
diaphragmatic protein synthesis. The extent to which pretranslational events contribute to
the MV-induced decrease in protein synthesis is unknown. Therefore, these experiments
were designed to determine the time course of changes in MHC mRNA after 6, 12, and
18 hours of MV. This was achieved by measuring the MHC mRNA content in
diaphragms from control and MV animals. Specifically, we measured the diaphragmatic
content of type I, IIa, IId/x, and IIb MHC mRNA in control animals and at the
completion of 6, 12, and 18 hours of MV.
Mechanical ventilation (MV) is an intervention used to sustain ventilation in
patients who are unable to maintain adequate alveolar ventilation. The withdrawal of MV
is commonly referred to as "weaning." Patients who experience weaning difficulties
commonly exhibit respiratory muscle weakness. Hence, it has been postulated that both
weakness and decreased endurance of respiratory muscles are major contributors to the
failure to wean patients from MV (167). This notion is strongly supported by recent
animal studies indicating that prolonged controlled MV results in significant reductions
in diaphragmatic force production. Further, our laboratory recently discovered that the
MV-induced diaphragmatic force deficit is associated with significant diaphragmatic.
History of Mechanical Ventilation
Galen (56), in the year AD 160, may have been the first to artificially ventilate an
animal. He reported that "If you take a dead animal and blow air through its larynx
(through a reed), you will fill its bronchi and watch its lungs attain the greatest
distention." More than 1000 years after Galen, Vesalius found that he could keep the
heart beating after a pneumothorax by inflating the lungs through a reed tied to the
trachea (171). In 1664 Hooke described dissecting a dog, putting a pipe into the trachea
and attaching the pipe to a bellows (15). The heart continued beating and the dog stayed
alive for over an hour (15).
Artificial ventilation of dogs led to the use of positive pressure ventilation to revive
human drowning victims in the mid 1700s (35). However, positive pressure ventilation
frequently caused fatal pneumothoraxes during animal experiments and was later
condemned by both the Academie Francaise and the Royal Humane Society (35).
Quashing positive pressure ventilation early in its development led to an alternative
method: negative pressure ventilation. Development and use of negative pressure
ventilation flourished in the 1800s and by 1928 the "Iron Lung" became the first negative
pressure ventilator used successfully on a large scale (35). The iron lung saved many
lives during the poliomyelitis epidemic in the 1930s and served as the mainstay of
treatment for respiratory paralysis from poliomyelitis until positive pressure ventilation
was reintroduced in the 1950s (35).
Positive pressure ventilation was heavily used in physiology laboratories during the
mid to late 1800s and by 1879 the volume-cycled ventilator was a common piece of
equipment at Harvard University (35). While positive pressure ventilation was used to
some degree before the 1950s, it was not until the poliomyelitis epidemic struck
Copenhagen in 1952 that its full utility was realized (35). Since then, positive pressure
ventilation has been used successfully to treat many medical conditions that lead to
In the 1980s a new method of positive pressure ventilation was introduced. This
new application was a noninvasive means of ventilation via a nasal, facemask, or an oral
connection and proved to be a significant development in MV (133). The evolution of the
mechanical ventilator has continued to this day. Its use as an indispensable life-saving
tool has insured its place in clinical practice.
Indication for Clinical Use of Mechanical Ventilation
MV is used for 4 main reasons:
Life support for a patient with a life threatening illness whose recovery is
Life support for a patient under general anesthesia during surgery.
To provide ventilation during respiratory muscle failure or to compensate for a
damaged upper airway.
As an aid during recovery or rehabilitation from an illness (3).
A common physiological outcome of these situations is respiratory failure. Respiratory
failure is often defined as a PaO2 of less than 50 mmHg at sea level while breathing a gas
mixture of at least 50% 02 and/or a PaCO2 greater than 50 mmHg hypercapniaa) (3).
Respiratory failure due to inadequate gas exchange is termed hypoxic respiratory
failure (134). If respiratory failure is due to ventilatory pump failure it is known as
hypercapnic respiratory failure, or the two may occur in combination (134). Hypoxic
respiratory failure is commonly associated with severe respiratory illnesses and can
induce hypoxemia by one or some combination of four mechanisms: alveolar
hypoventilation, right-to-left shunt in the heart, ventilation-perfusion mismatch, or
incomplete diffusion equilibrium (176).
The rib cage and its muscles, the diaphragm, and the abdomen and its muscles
make-up what is known as the ventilatory pump (160). Alveolar ventilation and gas
exchange depend on the ventilatory pump. Hypercapnia is a telltale sign of ventilatory
pump failure. A reduction in central neural drive, inspiratory muscle impairment, and/or
excessive respiratory workload can induce ventilatory pump failure (3). Neuromuscular
disease, drug overdose, or brainstem injury can impair central drive to the point of
inducing ventilatory pump failure (3). Inspiratory muscle performance can be negatively
impacted by neuromuscular disease (4), metabolic disturbances (3), certain drugs (2), a
disadvantageous length-tension relationship (175), mechanical disadvantage (103),
altered force-velocity relationship (3), detraining and atrophy (8, 92, 125), and fatigue (2,
135). Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary
resection for example, can also lead to respiratory pump failure.
Increased inspiratory muscle workload results in an increased work of breathing.
An increased work of breathing requires recruitment of the diaphragm (the primary
muscle of inspiration) and also recruitment of the accessory inspiratory muscles. This
presents two significant challenges: increased workload for the diaphragm and increased
oxygen consumption. The diaphragm is well suited to the constant demand of pulmonary
ventilation. However, if the constant workload exceeds 40% of its maximal force-
generating ability, it will fatigue (135) and ventilatory pump failure may ensue.
A further consideration of an increased work of breathing is the increased demand
for oxygen by the respiratory muscles. The respiratory muscles may account for more
than 50% of the total body oxygen consumption, as compared to less than 5% under
normal conditions (13). The increased oxygen cost of breathing reduces the availability
of oxygen to other body tissues and may lead to other potentially fatal events (e.g.,
myocardial ischemia) (167). As mentioned above, an increased work of breathing, if
excessive, may lead to diaphragmatic fatigue and thus hypoventilation. Hypoventilation
causes a loss of oxygen intake and also causes hypercapnia (which significantly impairs
muscle contractility) (85). This series of events, if left unchecked, can put the patient into
a downward spiral of respiratory muscle distress, hypoventilation, and hypercapnia (167).
This scenario may be remedied, however, by placing the patient on MV and thus
"resting" the diaphragm. In fact, mask ventilation typically improves respiratory
frequency, arterial oxygen tension, and pH soon after it is applied (6, 31).
Modes of Mechanical Ventilator Operation
The mode of MV depends on the needs of the patient (central drive, respiratory
muscle dysfunction, etc.). Controlled MV, assisted-control ventilation, intermittent
mandatory ventilation, and pressure support ventilation are all modes of MV commonly
used to aid the patient. The properties of each are outlined below.
Controlled Mechanical Ventilation
Controlled MV (CMV) is perhaps the most straightforward mode of MV.
Controlled MV delivers all breaths in a predetermined fashion. Breathing frequency (f),
tidal volume (VT), inspiratory-to-expiratory timing (I:E ratio), and inspiratory flow
pattern are each regulated by the ventilator settings. The patient's breathing frequency or
inspiratory effort cannot alter the preset respiratory parameters; hence, patient triggering
is not possible during CMV. Controlled MV is achieved pharmacologically (e.g.,
sedation and neuromuscular blockade) or by mechanical hyperventilation (78). The use of
CMV is limited to patients who are apneic because of brain damage, sedation, or
neuromuscular blocking agents (167). Controlled MV is used to treat hypoxemic
respiratory failure due to widespread atelectasis, localized alveolar disease,
noncardiogenic pulmonary edema, and cardiogenic pulmonary edema (3). Controlled MV
is also used to treat hypercapnic respiratory failure due to acute neuromuscular disease
and acute obstructive disease (3).
While CMV provides the maximum degree of respiratory muscle rest it is not
without consequence to the very muscle it is intended to aid, the diaphragm.
Administration of CMV often requires the use neuromuscular blocking agents whose use
is associated with prolonged weakness or paralysis lasting up to 1 week (68, 89, 143).
The effect of the neuromuscular blocking agents is compounded when corticosteroids are
given. The combination of neuromuscular blocking agents and large doses of
corticosteroids can result in generalized myopathy lasting weeks or months (44). Further,
this pharmacologic combination is associated with a higher incidence of muscle
weakness (93). Even in the absence of disease, CMV can induce muscle weakness which
is attributed to muscle atrophy (167).
Assist-control (AC) ventilation is the first mode of MV used in many institutions
(104). Assist-control ventilation provides a positive pressure breath in response to the
patient's inspiratory effort. The VT of each breath is set at the ventilator. The VT is
delivered with each inspiratory effort -or if the patient fails to trigger the ventilator
within a set amount of time. Assist-control allows the patient to control breathing
frequency. The patient also controls the ventilator-generated pressure. As the inspiratory
effort generated by the patient increases, the ventilator-generated assistance decreases
(104). Under the most favorable conditions, AC is 50-66% more effective than active
chest inflation at reducing respiratory work of breathing by (108).
Intermittent Mandatory Ventilation
Intermittent mandatory ventilation (IMV) provides a preset number of positive
pressure breaths and allows the patient to breathe spontaneously between ventilator-
delivered breaths. Intermittent mandatory ventilation can be set to provide a breath that is
either a preset volume or pressure. Once a predetermined pressure is reached, the
ventilator terminates the positive pressure breath. Further, IMV allows the patient to
autonomously alter his/her spontaneous breathing pattern. Because each IMV breath is
synchronized with the patient's breathing pattern, this mode of ventilation is also known
as synchronized intermittent mandatory ventilation (SIMV).
While IMV gives the clinician great flexibility in treating the patient, it has a
potential drawback. Intermittent mandatory ventilation was designed to provide volume
assistance while allowing the patient to breathe spontaneously (that is, to rest the
inspiratory muscles and attenuate inspiratory muscle deconditioning). However, when
IMV accounts for 20 to 50% of the total ventilation, the electromyographic (EMG)
activity of the diaphragm and the sternomastoid muscles is equal to that of spontaneous
breaths (81). While IMV is intended to provide inspiratory muscle rest, the EMG data
suggest that this may not be the case.
Pressure Support Ventilation
Pressure support ventilation (PSV) is designed to augment the patient's inspiratory
effort by providing positive pressure support. Pressure support ventilation reduces the
work of breathing by raising the airway pressure to a predetermined level after the patient
initiates a breath; and continues to do so until the end of the inspiratory effort is sensed as
a reduction in inspiratory flow (31). In PSV treatment, breathing frequency, VT, and
inspiratory flow pattern are determined by the patient. Pressure support ventilation is
widely used in intensive care units because it does not require heavy sedation of the
patient and it allows the patient to breathe spontaneously. Further, the patient is required
to use their inspiratory muscles, thereby reducing the severity of inspiratory muscle
Diaphragmatic Motion during Mechanical Ventilation
The diaphragm, like the lung, can be described in terms of its position relative to
the pressures acting on it. In the upright position there is a vertical gradient in pleural
pressure acting on the lungs, and the pressure acting on the upper portion of the lungs is
more subatmospheric than the pressure at the bases of the lungs. The region of the lungs
at the bottom of the vertical gradient is termed dependent and the region at the top of the
gradient is termed nondependent. Therefore, while in the upright position the bases of the
lungs are in the dependent region, and the apices are in the nondependent region. Shifting
the body position to the supine position changes the dependent and nondependent
relationships (i.e., the dorsal region of the lungs is in the dependent region and the ventral
surface of the lungs is in the nondependent region).
Likewise, while in a supine position, the ventral portion of the costal diaphragm is
in the nondependent position and the dorsal portion of the costal diaphragm and the crural
portion of the diaphragm are in the dependent region. The middle costal portion of the
diaphragm lies between both regions but is often considered to be dependent. While
breathing spontaneously in the supine position, the dependent diaphragm is displaced or
has a greater excursion than the nondependent diaphragm (54, 151, 87) because of
anatomical differences between the costal and crural diaphragm region (87).
After anesthesia and MV, the position of the diaphragm at functional reserve
capacity (FRC) shifts cephalad (23, 54, 87, 129). The cephalad shift is due to loss of
muscle tone in the diaphragm and gravitational displacement of the abdominal contents
(23, 54, 129). After CMV the pattern of displacement is reversed: the dependent regions
of the diaphragm are displaced less than the nondependent regions of the diaphragm (54).
This reversal of displacement is the result of a uniform increase in thoracic pressure
displacing the diaphragm where abdominal pressure is least (i.e., the nondependent
region of the diaphragm) (54). Thus, during MV the diaphragm is passively moved each
time a breath is artificially delivered to the patient.
The diaphragm does not shorten to the same extent during MV as during
spontaneous breathing. The diaphragm does, however, shorten passively while being
displaced by the artificially ventilated lungs (119). The degree to which the diaphragm
passively shortens during MV is not uniform. Diaphragmatic shortening during PSV (87,
129) and CMV (151) has been reported to be less than that of spontaneous breathing.
However, others (53, 132) have reported greater diaphragmatic shortening during CMV
than during spontaneous breathing (119).
The disparity between these findings may be due to differences in methodology.
The studies that reported less diaphragmatic shortening during MV used indirect methods
such as 3-dimensional x-ray tomography (87), CT scans (129), and videofluoroscopy
(151) to measure diaphragmatic length. The studies that reported greater diaphragmatic
shortening during MV used Sonomicrometry (119). Sonomicrometry is a more accurate
and perhaps more reliable method for measuring muscle length changes. Despite the
different length changes reported during MV, the unifying finding is that the diaphragm
shortens passively. In addition to passive shortening, the diaphragm is also displaced by
the lungs during MV.
Weaning from Mechanical Ventilation
The common term for discontinuation of MV is "weaning." This term refers to the
slow withdrawal of MV at a rate the patient can tolerate. The weaning success rate in
many intensive care units (ICU) is usually higher than 70% depending on the subset of
patients (94). A person is considered a "weaner" if he/she is breathing spontaneously 2
days after discontinuation of MV (94). A patient who requires some degree of ventilatory
support (total or partial) is considered a "non-weaner" (94).
It is imperative to discontinue use of MV as soon as possible because MV is
associated with several major complications. Most patients requiring short-term MV
experience little difficulty when MV is withdrawn. As previously discussed, MV is
frequently used to aid patients recovering from respiratory failure. Discontinuing MV for
many of these patients is difficult. Because of the factors that led to the patients'
placement on MV, great care is given to their discontinuation from MV. The process of
discontinuation from MV is challenging and makes up a large portion of the ICU
The process of weaning may require more than 2 days and it can become a lengthy
process. "A long-term ventilator-assisted individual is a person who requires mechanical
ventilatory assistance for more than 6 hours a day for more than 3 weeks after all acute
illnesses have been maximally treated and in whom multiple weaning attempts by an
experienced respiratory care team have been made" (105). While MV may not be the
primary reason a patient is in the hospital, it is often the reason for a prolonged stay. The
total number of difficult weaners ranges from 20% up to 70% in some ICUs (94) and the
cost to treat these patients is large (105). A 1983 study estimated that there were 6,800
long-term MV patients at a cost of $1.7 billion per year or -1.5% of total hospital costs
(106). In 1990, the American Association for Respiratory Care commissioned a rigorous
study of the incidence and cost of long-term MV care (7). This study found that there
were 11,419 long-term MV patients at an annual cost of $3.2 billion for treatment (7).
While these studies relay the magnitude and dollar cost of MV, the importance of the loss
of individual independence as well as the emotional cost should not be forgotten.
Properties of the Diaphragm
The diaphragm is the primary muscle of inspiration. As such, it is chronically
active and its metabolic characteristics reflect this. This section reviews the functional
and metabolic characteristics of the diaphragm.
Function of the Diaphragm
Breathing has long been recognized as a vital process. For example, around 2000
BC, Chinese philosophers wrote about "lien ch'i," the process of bringing the inspired
breath into the soul substance (35). The ancient Greeks also believed that breathing was
essential and that the diaphragm was the seat of the soul (102). The Greek word phrenes
means soul; this is how the phrenic nerves were named (102). In the third century BC, the
diaphragm was recognized to be a muscle by Erasistratus and he taught that it was the
main muscle of inspiration (43).
The diaphragm is chronically active skeletal muscle and is innervated by the
phrenic nerves from the cervical segments 3, 4, and 5 (176). The diaphragm has two
functionally different and distinct parts: the central tendon (the non contractile portion)
and the costal and crural regions (the muscular portions). The costal and crural muscle
fibers extend outward from the central tendon. The fibers of the crural diaphragm radiate
from the central tendon and insert onto the anterolateral aspect of the first three lumbar
vertebrae and the aponeurotic arcuate ligaments (39). The fibers of the costal diaphragm
extend from the central tendon and insert on the xyphoid process of the sternum (the
ventral region) and the upper margins of the lower 6 ribs (the medial and dorsal regions)
(39). The muscle fibers of the costal diaphragm run cranially from their insertions and are
thus apposed directly to the inner aspect of the lower rib cage (39).
Chest wall displacement during inspiration is accomplished by the unique shape
and location of the diaphragm. The healthy diaphragm is an elliptical cylinder capped by
a dome (39). The dome region is primarily composed of the central tendon. The
cylindrical region is the portion apposed directly to the inner aspect of the lower rib cage,
the "zone of apposition" (39). This shape and location gives the diaphragm the ability to
increase chest wall dimensions and therefore inflate the lungs (123).
Activation of the diaphragm elicits a caudal force onto the central tendon and a
cephalic force onto the lower 6 ribs by the costal diaphragm and vertebral column by the
crural diaphragm (123). The caudal force causes the dome of the diaphragm to descend
and displace the abdominal contents downward, but the abdominal contents resist
displacement and therefore act as a fulcrum (160). Thus, as the diaphragm contracts and
its fibers shorten, the transverse dimensions of the chest wall increase (123, 160).
Contraction of the diaphragm also increases the cephalo-caudal dimensions of the chest
wall (123). Based on the insertions of the costal and crural diaphragm, the chest wall
dimensions are changed by the costal region as the crural region inserts onto the
immovable vertebral column and only displaces the abdomen (123, 160).
Metabolic Characteristics of the Diaphragm
The anatomical and morphological design of the diaphragm is well suited to the
constant demand of the ventilatory system. Likewise, the metabolic design of the
diaphragm allows it to meet the constant challenge imposed on it.
Skeletal Muscle Fiber Types within the Diaphragm
Skeletal muscle is a highly plastic tissue (it has the ability to adapt to the workload
imposed on it). Skeletal muscle meets the workload by expressing fiber types best suited
to the demand. Thus, the heterogeneity of skeletal muscle fibers expressed within a
muscle is a reflection of the "job" the muscle is responsible for. The diaphragm is a
highly specialized skeletal muscle. It is the only skeletal muscle that is chronically active
for life and its fiber type expression reflects this.
Fiber typing has evolved a great deal beyond the initial classification of "red" and
"white" put forth by Ranvier in 1873 (128). As reviewed by Pette and Staron (120)
muscle fibers can be classified by various methods including histochemistry,
immunohistochemistry, and gel electrophoresis.
Histochemical classification is a subjective method based on myofibrillar
actomyosin adenosine triphosphatase (ATPase) activity or aerobic and anaerobic
metabolic enzymes. These methods typically reveal 3 fiber types: I, IIa, and IIb (via
ATPase) or slow-twitch oxidative, fast-twitch oxidative glycolytic, and fast-twitch
glycolytic (via enzymatic analysis) (120). Immunohistochemistry and gel electrophoresis
are able to resolve 4 fiber types based on myosin heavy chain (MHC) proteins: I, IIa,
IId/x, and IIb. Immunohistochemistry and gel electrophoresis have been used to
determine the fiber type composition of rat and human diaphragm (Table 2-1). Overall
however, the human and the rat diaphragm are very similar in fiber type composition
Recently, the MHC content of human single muscle fibers were thoroughly
characterized histologically, immunohistologically, and electrophoretically (46, 140).
This information was then correlated with the mRNA transcripts for each MHC gene.
Table 2-1. Fiber type composition (%) of the diaphragm and locomotor skeletal muscles.
All values reported are percentages. Because of rounding, values may not total 100%.
DIA = whole diaphragm. COD = costal diaphragm. PL, plantaris; SOL, soleus; VL,
vastus lateralis; HC, histochemistry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis; IH, immunohistochemistry.
The mRNA transcripts in all fibers classified as lib using conventional methods are
actually equivalent to the rat IIx gene (46, 140). Based on these findings it was suggested
that all type II fibers previously identified as lib would be more accurately classified as
IIx (46, 140). Additionally, conventional histochemical techniques may have erroneously
classified rat IIx fibers as lib (120). In light of these findings, the diaphragms from the rat
and human are quite similar, as neither has many type lib fibers.
The oxidative capacity of skeletal muscle is consistent with its function. Skeletal
muscles that contract tonically are slow twitch and highly oxidative, whereas muscles
used sporadically are fast twitch and have a low oxidative capacity. The characteristics of
the diaphragm fit this biochemical tenet. Most muscle fibers in the diaphragm are highly
oxidative and this is in-line with the predominant fiber types in the diaphragm, type I and
Typical biochemical markers of skeletal muscle metabolic pathways include citrate
synthase (CS), succinate dehydrogenase (SDH), 3-hydroxyacyl-CoA dehydrogenase
(HADH), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase
(LDH). The oxidative capacity of a muscle is often estimated by the citric acid cycle
enzymes CS and SDH. Likewise, the lipolytic capacity can be determined by an integral
P-oxidation enzyme, HADH. The enzymes HK, PFK and LDH are often used to
determine the glycolytic capacity of muscle cells
To date, there are two published reports of normal human diaphragmatic
bioenergetic enzyme capacities (139, 163). Sanchez et al. (139) compared the
bioenergetic capacity of the diaphragm to the latissimus dorsi. In each case,
thebioenergetic capacity of the diaphragm was significantly greater than that of the
latissimus dorsi (i.e., CS 180%, HADH 215%, HK 170%, and LDH 115%).
Table 2-2 shows the bioenergetic similarities of the human and rat costal
diaphragm, as well as two different locomotor muscles. Note that the bioenergetic
capacity of the human and rat diaphragm are very similar. Overall, the bioenergetic
capacity of the diaphragm reflects its continuous use, a high oxidative capacity as
measured by citric acid cycle and P-oxidation enzymes, and a moderate glycolytic
Table 2-2. Bioenergetic enzyme activities in the costal diaphragm and two locomotor
Muscle CS HADH LDH Reference
Human COD 0.33 0.27 11.6 (163)
Rat COD (Rat) 0.46 0.23 4.8 (126)
PLA (Rat) 0.29 0.11 8.4 (126)
SOL (Rat) 0.33 0.22 2.5 (126)
COD, costal diaphragm; PL, plantaris; SOL, soleus; CS, citrate synthase; HADH, 3-
hydroxyacyl-CoA dehydrogenase; LDH, lactate dehydrogenase. Enzyme activities are
expressed as jiM/min/mg of protein.
The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly
adapts to the demands placed on it. Skeletal muscle quickly adapts to alterations in
proprioceptive activity, motor innervation, mechanical load, and joint mobility (10).
Skeletal muscle adapts to an increase in muscular activity by increasing contractile and
structural protein content (hypertrophy), whereas inactivity or disuse leads to a loss of
muscle mass (atrophy) (10). Hypertrophy (protein accumulation) and atrophy (net loss of
protein), therefore, are critically dependent on the relative rates of protein synthesis and
protein degradation (61). Atrophy results in a decrease in cross sectional area (CSA) and
this is functionally significant because muscle strength is directly related to CSA (26).
MV is a common method of reducing or removing diaphragmatic work. The
reduction in workload by MV leads to diaphragmatic disuse muscle atrophy and/or
weakness and is a major mechanism of weaning failure (167). The term disuse is relative
and can be defined as a reduced level of contractile activity (116). Two characteristic
components of reduced contractile activity include hypokinesia and hypodynamia (116).
Hypokinesia refers to a decreased level of contractile activity (i.e., reduced limb
movements) and hypodynamia is a decrease in mechanical loading (i.e., reduced weight-
bearing function) (116). MV and other models of skeletal muscle disuse atrophy are
Models of Locomotor Muscle Atrophy
Several different experimental models and human clinical conditions result in
skeletal muscle atrophy. These models and conditions will be reviewed according to the
level of neuromuscular activation altered by electrical activation and weight-bearing
Reduced Electrical Activation and Load Bearing
The electrical activation and load bearing status of skeletal muscle can be altered
by spinal cord injury, spinal cord transaction, and limb immobilization (with the muscle
of interest in the shortened position). In this section, spinal cord injury (SCI) and spinal
cord transaction (ST) will be considered together. Spinal cord transaction interrupts the
upper motor neuron pathway by transecting the spinal cord, often at the thoracic level. In
cat soleus, ST results in a 75% reduction in the daily-integrated electromyographic
(EMG) activity and a 66% reduction in the total duration of the EMG (1). After ST, the
ankle joint of the animal is held in an extended position, effectively unloading the soleus
(1). After 5 and 10 days of ST, the rat soleus significantly atrophies, -35% and 60%
reduction in the total CSA respectively (45). Further, ST alters myosin expression from
slow-to-fast MHC at both the mRNA (45) and protein level in the soleus (45, 138). Six to
8 months after ST biochemical measures such as citrate synthase and myosin ATPase
activities become characteristic of fast muscle (138). The functional outcome of ST is a
loss of absolute force generating ability by the affected muscless. The absolute tension
generation by the medial gastrocnemius and soleus after 6 to 8 months of ST is reduced
by 26% (137) and 50% respectively (138).
Human skeletal muscle also undergoes a slow-to-fast MHC shift After SCI. The
vastus lateralis from 15 patients was sampled 6, 11, and 24 weeks after SCI (33). At the 6
and 11 week time point there was no detectable MHC shift but after 24 weeks there was a
16% increase in type IIx MHC expression (33). Thus, human skeletal muscle adapts to
reduced electrical activation and muscle loading but at a slower rate than small animals
such as rat and cat. Overall, ST and SCI lead to alterations in fiber type composition and
impairs force generation.
Hindlimb immobilization (HI) is a clinically relevant model of reduced electrical
activity and loading. Hindlimb immobilization is achieved by fixing the joint(s) at a
specific angle by either pinning the joint(s) surgically with steel rods or by fixing the
limb with orthopedic plaster. The electrical activation of the rat soleus after HI in the
neutral position is -50% of control (52). Similarly, the electrical activity of the human
quadriceps is -60% less than the electrical activity observed in control quadriceps (77).
Atrophy induced by HI results in significant decrements in the CSA of both rat (9) and
human (77) skeletal muscle within 7 to 21 days, respectively. Accordingly, the decrease
in CSA results in an -50% loss of strength in rat soleus (177) and human quadriceps (77)
skeletal muscle. Metabolic markers such as citrate synthase and lactate dehydrogenase
indicate that HI shifts the normally slow oxidative soleus toward a fast oxidative
glycolytic muscle (51). Further, the slow-to-fast MHC shift occurs rapidly at the mRNA
level. After 1 week of HI soleus IIx and IIb mRNA transcript levels increased -24 and
2.6 fold compared to control (83). Fiber type conversion in human skeletal muscle After
HI also follows the slow-to-fast pattern. After 3 weeks of HI, type I mRNA transcripts
were -30% less than control and IIx transcripts were -300 times greater than control
(77). To date, there are no data on the levels of MHC protein isoforms after HI in either
the rat or humans.
Within hours of HI, protein synthesis and protein degradation are altered. Six hours
after HI the fractional rate of protein synthesis in the soleus is reduced 20-35% (28, 60).
For example, Goldspink (60) measured a 20% decrease in the synthetic rate with -2%
loss of muscle wet weight and a slight decrease in the rate of protein breakdown after 6
hours of HI. After 2 days of HI, soleus wet weight was reduced by 25%, the rate of
protein synthesis was 65% less, and the rate of protein degradation was 50% greater than
control (60). Booth (28) reported similar findings after 6 hours of bilateral HI, a -35%
decrement in the fractional rate of protein synthesis in the gastrocnemius.
Due to the limited nature of spaceflight, ground based models such as hindlimb
unloading (HU) of the rat, and human bed-rest models have been devised as models of
Hindlimb unloading of the rat is typically achieved by placing a plaster cast at the
base of the tail to which a gimbal is attached. The animal is then raised such that the
forelimbs support the weight of the animal and its hindlimbs are not in contact with the
ground. To date, EMG recordings of rat hindlimb activity have not been made during
actual spaceflight. However, EMG recordings of rat soleus during parabolic flight, -25
sec of micro-gravity, is -10% of control values (95). This rapid decrease in EMG activity
has also been observed using HU. The EMG activity of the soleus is nearly abolished -3
sec after HU is imposed (5). Chronic soleus EMG activity either remains decreased (24)
or returns to normal (5) after 28 days of HU. While actual force measurements have not
been made during spaceflight or HU, it is generally accepted that force generation is
minimal (159). The contractions are spontaneous isotonic contractions and do not
generate much force because they are not weight bearing and are similar to the contractile
patterns that occur in the muscles of astronauts in space (27).
Spaceflight and HU rapidly affect muscle weight. After 5 to 7 days of spaceflight,
rat soleus mass is -18 to 30% less than control (75). Additionally, soleus muscle fiber
CSA is significantly reduced (-14%) and absolute tension is significantly impaired (-
28%) (75). Hindlimb unloading induces similar outcomes after 4 to 5 days, -15-30% loss
of soleus mass (101, 155). Seven days of HU significantly reduces type I fiber CSA, -
14%, and absolute tension, -30% (110). Lactate dehydrogenase activity is not altered by
spaceflight, but HU significantly decreases its activity, -27% (117). Conversely, citrate
synthase activity is not affected by either spaceflight or HU (117). While the unloading
effect significantly alters slow antigravity muscles such as the soleus, the morphometry
and function of the fast extensor digitorum longus is not impacted by 14 days of
The change in MHC phenotype in the HU soleus is a very predictable slow-to-fast
shift. After 4 days the loss of type I MHC is small, but after 7 days the loss is significant
(155). The increase in type IIa MHC expression is significant after only 4 days (155).
After 7 days, type IId/x MHC is significantly increased and an increase in type IIb is
found after 14 days (155). The expression of each MHC mRNA transcript roughly
mirrors protein expression. A small change in type IP3 MHC mRNA (the mRNA
transcript for type I MHC) expression occurs after 4 to 7 days and a significant increase
in the expression of Icl mRNA (the transition mRNA transcript from I3 to IIa) occurs in
the same time period (155). Type IIa mRNA expression increases slightly and IId/x and
IIb expression significantly increases within 4 days (155). The decrease in type I and IIa
mRNA (>30%) and the increase in IId/x and IIb (>100%) mRNA becomes significant
after longer periods of time, 9-14 days in space (32, 67) and thus, the slow-to-fast MHC
shift rapidly occurs at both the protein and mRNA level.
A contributory factor leading to the loss of muscle mass under reduced loading
conditions is the decreased rate of protein synthesis. Total mixed protein synthesis and
myofibrillar protein synthesis measured during the first 5 hours of HU decreases 16% and
22% respectively in the rat soleus (162). In these experiments protein synthesis was
measured by constant infusion of radio-labeled leucine into the animal over the 5 hour
period. The measurement of protein synthesis during the first 5 hours of HU was
therefore an average of the initial response and may not reflect the true rate during the 5th
hour. However, after 24 hours of HU, total and myofibrillar protein synthesis in the
soleus is significantly diminished, -30 and -15% respectively (115). Thus, skeletal muscle
adapts rapidly to HU by decreasing protein synthesis and importantly, the rate of
myofibrillar protein synthesis.
Models of Inactivity
Inactivity includes spinal cord isolation and blockage of the motoneuron action
potential conduction by substances such as tetrodotoxin. Also,denervation is often
included as a model of inactivity. Denervation is achieved by severing the motoneuron-
muscle connection, but because it also interrupts neural input to tissues such as vascular
tissue, thus altering blood flow, its effect is often difficult to interpret (116) and will not
be considered here.
Spinal cord isolation (SI) has been used to study the effects of nueromuscular
activity on skeletal muscle. Spinal cord isolation involves complete spinal cord
transaction at the mid or low thoracic and lumbar-sacral levels, as well as complete
deafferentiation between the two points of transaction (159). This preparation maintains
the integrity of the neuromuscular unit and yet eliminates sensory input from the dorsal
roots as well as neural signals from either above or below the transaction (159).
The atrophic effect of SI is severe and rapid. The soleus loses 25% of its mass and
75% of its fiber CSA 4 days after SI (64). The slow-to-fast shift in MHC is relatively
slow, resulting in a 10% increase in IId/x expression after 15 days and a 40% loss of type
I MHC after 60 days (64). Spinal cord isolation causes skeletal muscle to become not
only smaller and faster, but weaker as well. Maximum force generation by the soleus 6
months after SI is decreased by 80% (136). The enzymatic profile of skeletal muscle
shifts from oxidative (-70% succinate dehydrogenase activity) toward glycolytic (+120%
glycerolphosphate dehydrogenase activity) after SI (84). To date, there are no published
reports of total and/or myofibrillar protein synthesis After SI, however, the rate and
severity of SI- induced atrophy suggests that both would be decreased.
Tetrodotoxin (TTX) is a potent Na+ channel blocker and if applied continually to
motor nerves it will inactivate skeletal muscle. Muscle paralysis after TTX treatment, as
determined by EMG activity during locomotion, is nearly complete in all treated animals
(152). This model of inactivity leads to significant atrophy, 46.5% decrease in muscle
mass and a 25% decrease in myofibrillar protein in 2 weeks (152). Concomitant with the
TTX-induced atrophy is a 38% loss of force generating ability (152). The expression of
MHC is altered by TTX treatment such that the expression of type I MHC increases, type
IIa decreases, type IId/x increases, and type IIb does not change (112). It is currently
unknown why type I MHC expression increases in response to TTX treatment but it may
be related to the disruption in the delivery of a neurotrophic factor to the motor end-plate
(159). Tetrodotoxin treatment severely diminishes the metabolic capacity of both
oxidative (citrate synthase -33%) and glycolytic (phosphfructokinase -70% and ca-
glycerolphosphate dehydrogenase -58%) enzymes (153). The effect of TTX treatment on
protein synthesis is currently unknown but it likely plays a role in the observed atrophy
Models of Atrophy in the Diaphragm
The primary muscle of inspiration is the diaphragm and it is chronically active
throughout life. Due to its chronic activity the activation pattern of the diaphragm differs
from locomotor muscles such as the extensor digitorum longus (EDL) and the soleus. The
rat diaphragm has a duty cycle (duration of inspiratory time/total respiratory cycle
duration) of -40% (141) while the EDL and soleus have duty cycles of 2% and 14%
respectively (71). Thus, the contractile history of the diaphragm differs greatly from
locomotor muscles and it may be more susceptible to alterations caused by inactivity
(113). Models of diaphragmatic inactivity include denervation, blockade of nerve
impulses by TTX, spinal cord transaction (ST), and MV. The effects of MV will be
considered separately from the other models.
Procedure for Investigating Diaphragmatic Atrophy
Diaphragmatic denervation (DNV) is accomplished by dissecting the phrenic
nerve, unilaterally or bilaterally. Once dissected, the phrenic nerve is transected
(phrenicectomy) and a significant portion is removed (e.g. -10-20 mm in the rat) to
prevent possible reinnervation of the diaphragm (98).
Again, TTX is a Na+ channel blocker that prevents action potential propagation.
The phrenic nerve is typically dissected at the lower neck and a Silastic cuff is placed
around the nerve and connected to a miniosmotic pump that continuously perfuses the
nerve with TTX.
Spinal cord transaction (ST) is achieved by performing a dorsal laminectomy after
which one-half (e.g. right side) of the cervical spinal cord (e.g. at C2) is sectioned from
the dorsal root to the ventral root. Correctly done, only the ventral and lateral funiculi are
cut and the lateral funiculus is preserved in order to minimize motor deficits in the
ipsilateral side (113).
Paralysis of the diaphragm is verified by the EMG activity of the left and right
sides (hemidiaphragm) of the diaphragm. The EMG signal is obtained by implanting
small diameter wire electrodes into the diaphragm. Each of the above-mentioned surgical
preparations induces diaphragmatic paralysis (i.e., no EMG activity) of the intended
hemidiaphragm, and a 50% increase in the activity of the intact hemidiaphragm due to a
compensatory increase in muscle activation (183). However, phrenic nerve activity after
unilateral DNV and unilateral TTX significantly increases -40-50% on both sides (113).
The increase in activity indicates that there is a compensatory increase in central drive to
motor neurons on both sides of the spinal cord (113). The ST model, however, leads to
inactivity of the phrenic motoneurons (113). Diaphragm paralysis and motoneuron
activity are matched in the ST model, whereas motoneuron activity and paralysis are not
matched in the TTX model, and the connection between the nerve and muscle is
obviously severed in the DNV model (113). The morphological, biochemical, and
mechanical alterations that result from each of the above models is attenuated when the
activity of the diaphragm muscle fibers is matched by the phrenic motoneuron.
The initial response to diaphragmatic paralysis via DNV is hypertrophic after
which the response becomes atrophic. After 8 days of unilateral and bilateral DNV, the
diaphragm hypertrophies 20% (paralyzed hemidiaphragm) and 13% (each
hemidiaphragm hypertrophied) respectively (179). The hypertrophic response is
diminished by the second week and diaphragmatic mass returns normal (185). The
chronic response (i.e., 6 weeks) to diaphragmatic paralysis is a significant loss of
diaphragmatic mass (-37%) (98). To date, the effect of ST and TTX on diaphragmatic
mass has not been determined.
Differential Fiber Type Response to Diaphragmatic Inactivity
The change in cross-sectional area (CSA) after unilateral hemidiaphragm paralysis
induced by ST, TTX, and DNV is variable; in each case the alterations brought about by
ST are not as dramatic as those of TTX and DNV after 2 weeks of treatment. Type I fiber
area hypertorphies 33, 70 and 80% according to treatment, ST, TTX, and DNV,
respectively (184). Type IIa fibers also hypertrophy 13, 99, and 81% after ST, TTX, and
DNV respectively, (184). Paralysis induced by TTX and DNV leads to the appearance of
hybrid type I/IIa fibers (184). Type IId/x fiber CSA is reduced 5, 34, and 39% and type
IIb fibers also atrophy 15, 53, and 57% after 2 weeks of ST, TTX, and DNV respectively
(184). The variable response to the treatments may be the result of better matching
between muscle fiber and motoneuron activity in the ST model. After 6 weeks of
unilateral DNV, the CSA of the type I and IIa fibers returns to normal, whereas the IIb/x
fibers lose -57% of their CSA (98). The hybrid I/IIa fibers that appear after 2 weeks of
DNV (184) occupy -50% of the total CSA after 6 weeks of DNV (98), indicating a slow-
to-fast shift in MHC.
Myosin Heavy Chain Content
Diaphragmatic MHC content has been followed over a 3-week period of DNV
(147). Type I MHC increases after 1 week (+30%) and begins to decrease during the 2nd
(+25%) and 3rd week (-6%) (147). The type IIa MHC expression is elevated during the
first 2 weeks, +23% week one and +29% week two, and begins to return to normal by the
3rd week, +11% (147). The expression of type IId/x MHC falls off rapidly during the
first 2 weeks, -27% and -24%, and then begins to return to normal by week three, -6%
(147). Type IIb MHC expression declines rapidly and remains depressed over the entire
3-week period, -50% to -66% (147).
Myosin Heavy Chain mRNA
Northern analysis of MHC mRNA reveals a down regulation of all 4 MHC
transcripts after 8 days of DNV (179). Type I, IIa, IId/x, and IIb mRNA levels
significantly decrease 50, 70, 60, and 35% respectively after 8 days of reduced activity
(179). This is an interesting observation when one considers the finding that type I and
IIa MHC content increases and IId/x and IIb expression decreases at the protein level
(147). These observations suggest differential posttranscriptional regulation of the four
After 2 weeks of unilateral hemidiaphragm paralysis induced by ST, TTX and
DNV, specific tension declines 23, 49, and 51% respectively, (113). In accordance with
the observed, type I and IIa hypertrophy (184) and the increase in type I and IIa MHC
expression after diaphragmatic paralysis, diaphragmatic contraction and relaxation times
dramatically increase (113). Additionally, actomyosin ATPase activity is reduced by all
three treatments (184). Again, the effects of ST were not as pronounced as those induced
by TTX or DNV (113, 184).
Succinate dehydrogenase (SDH), a marker of oxidative capacity, is significantly
reduced after 2 weeks of ST, TTX and DNV (184). The reduction in SDH activity did not
occur in all fiber types however. The oxidative capacity was not altered in the type I or
IIa fibers. Paralysis induced by TTX and DNV reduced SDH activity in type IId/x and IIb
fibers whereas ST only reduced SDH activity in the IId/x fibers (184).
To date, measures of protein synthesis have only been performed using the DNV
model of disuse. As might be expected, in vivo protein synthesis is significantly increased
(-50%) during the initial hypertrophic phase, days 1 through 10 (169). The significant
increase in protein synthesis after acute DNV has also been observed under in vitro
conditions (170). However, after approximately 3 weeks of DNV, the diaphragm
atrophies significantly (29). While the rate of protein synthesis has not been measured
after chronic diaphragm DNV, measures of diaphragmatic total RNA content have been
made. During the hypertrophic period as well as the atrophic period, followed out for 51
days, total RNA content in the diaphragm parallels diaphragmatic mass (29). The
decrease in total RNA content and the loss of diaphragmatic mass suggests that protein
synthesis would be suppressed.
Diaphragm Length Changes
It has been hypothesized that passive stretch is an underlying mechanism
responsible for the morphological adaptations incurred by the paralyzed side of the
diaphragm (183). The paralyzed hemidiaphragm is "pulled" along by the intact
hemidiaphragm as it contracts. Due to differences in muscle fiber orientation, the length
changes that occur after unilateral paralysis varies between regions of the diaphragm. The
muscle fibers in the paralyzed midcostal region of the diaphragm are stretched -3-5%
beyond resting length (diaphragm muscle fiber length at end expiration) because they are
in series with the muscle fibers in the intact hemidiaphragm. The sternal region is
passively shortened 4-5% of resting length because the muscle fibers are in parallel with
the fibers of the opposite side (183). However, the degree of adaptation, as measured by
in vitro contractile properties and fiber typing, was similar between regions, costal and
sternal. If alteration of muscle fiber length, stretching or shortening, was the underlying
cause for the observed changes in in vitro contractile properties after DNV, a differential
response (e.g., passive shortening would induce greater contractile dysfunction) would be
predicted. This was not the case and the authors suggest that removal of innervation itself
is the underlying mechanism leading to the morphological adaptations (183). This
observation is supported by earlier findings that reported less pronounced morphological
and contractile adaptations after ST as compared to TTX or DNV (184). This suggests
that interactions between the motoneuron and muscle fibers may play an integral role in
muscle adaptation (183).
Mechanical Ventilation and Diaphragmatic Atrophy
MV is used to either fully support or augment alveolar ventilation. Further, MV
"rests" the diaphragm and in doing so the phasic activity of the diaphragm is decreased
by varying degrees, depending on the level of mechanical support.
Inhibition of diaphragmatic EMG activity during controlled MV (CMV) has been
demonstrated in healthy subjects (91, 107, 148, 149) and in chronic obstructive
pulmonary disease (COPD) patients (30). Likewise, MV reduces neuronal activity in
regions of the brain that are known to be involved in the control of breathing (48).
Importantly, the loss or reduction of EMG activity of a muscle is a primary factor in the
etiology of disuse atrophy (e.g., 45, 52).
Diaphragmatic Disuse Atrophy
Mechanical ventilation is frequently used in caring for neonates and infants. The
CSA of muscle fibers from the diaphragms of neonates and infants ventilated for more
than 12 days before death is markedly smaller, -70% in one case, and consistent with
disuse atrophy (86). Diaphragmatic atrophy may also be observed after cervical fracture.
Patients with lesions above the origins of the phrenic nerve roots require ventilatory
assistance (e.g., MV or phrenic nerve pacing). Measures of diaphragm thickness have
been performed on patients treated with phrenic nerve pacing. The normal adult
diaphragm is -0.3 cm thick (109) and after 8 months of MV the thickness can decrease to
-0.18 cm (14). After 6 weeks of phrenic nerve pacing, diaphragmatic thickness can
increase nearly two-fold with a three-fold improvement in maximal tidal volume (14).
While this is not conclusive evidence of diaphragmatic atrophy induced by prolonged
MV, it is highly suggestive of diaphragmatic atrophy.
Animal Models and Mechanical Ventilation
Due to the invasiveness of intubation and diaphragmatic biopsies, animal models
have been devised to study the mechanical and biochemical effects of MV on the
diaphragm. Anzueto et al. (8) tested the hypothesis that prolonged MV would impair
diaphragmatic contractile properties. The investigators ventilated healthy adult baboons
for 11 days and measured maximal transdiaphragmatic pressure (Pdimax), the force
frequency response, and endurance time pre- and post-MV (8). The ability of the
diaphragm to generate force in vivo can be determined by measuring Pdimax. This
measurement of diaphragmatic strength was impaired by 11 days of MV, -32% to -48%.
The diaphragmatic response to phrenic nerve stimulation over a wide range of
frequencies (i.e., the force frequency response) was dramatically shifted downward after
11 days of MV (8), indicating a loss of force generating ability across a spectrum of
stimulation frequencies. Fatigue resistance was assessed by requiring the animals to
breath through a resistor at 60-70% of Pdimax until the target pressure could no longer be
met. Diaphragmatic endurance time was reduced by -36% after 11 days of MV (8).
It is possible that the use of a neuromuscular blocking agent during the MV period
contributed to the deleterious effects of MV. The investigators did however control for
this by withholding the neuromuscular blocking agent for 8 hours before contractile
measurements were made. Contractile measurements were conducted only after
spontaneous breathing resumed and the diaphragm responded to phrenic nerve
stimulation (8). Typically, muscle weakness occurs when neuromuscular blocking agents
are used in conjunction with corticosteroids (36, 93) and only rarely occurs in patients not
treated concurrently with corticosteroids (59). Nonetheless, the decrease in diaphragmatic
force generation is similar to the results of Le Bourdelles et al. (92) and Powers et al.
(125) who did not use neuromuscular blocking agents to study the effects of MV.
The rat has also been used to investigate the impact of MV on the diaphragm. After
48 hours of MV, the mechanical properties of the diaphragm, soleus, and EDL as well as
their biochemical properties have been documented (92). MV did not significantly alter
body mass but the mass of the diaphragm, soleus, and EDL were significantly decreased
after 48 hours of MV (92). MV did not affect the contractile properties of the soleus or
EDL (92). However, diaphragmatic contractility was significantly impaired by 48 hours
of MV. MV resulted in a downward shift in the force frequency response and reduced
maximal tetanic tension -60% (92). Further, MV negatively impacts indicators of
calcium handling within the muscle such as the rate of force development (dP/dt) and the
rate of relaxation (92). The slowing of these indicators suggests a decreased rate of
calcium release and sequestration. MV did not affect the bioenergetic enzymes, citrate
synthase and lactate dehydrogenase in the diaphragm (92). This indicates that the
observed diaphragmatic dysfunction is not necessarily due to a derangement of
metabolism but rather, an alteration in one or more steps of excitation-contraction
Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties
Recently, our laboratory characterized the time course of MV induced
diaphragmatic dysfunction (125). Animals were mechanically ventilated in the control
mode for 12 to 24 hours. At the specified time point, a segment of the costal portion of
the diaphragm was carefully dissected and used for in vitro contractile measurements.
The body mass of each animal was measured pre- and post-MV and there was no change
(p > 0.05). Additionally, the mass of the soleus did not differ between control and MV
animals (p > 0.05). Arterial blood pressure, blood gases, and pH status were monitored
throughout the MV period and were found to fluctuate, but remained within a narrow
physiological range. Maintenance of blood gas and pH homeostasis is critical because
hypoxia, hypercapnia, and respiratory acidosis are known to impair diaphragmatic
Our findings reveal that contractile dysfunction occurs in as few 12 hours. Twitch
tension was 35% less than control after 12 hours of MV. Twitch tension continued to fall
over the 24-hour period; at which point, the twitch tension of the mechanically ventilated
diaphragms was 42% less than control. The force frequency response of the mechanically
ventilated diaphragms was impaired, shifted to the right, at all stimulation frequencies.
The magnitude of the right-shift was exacerbated with time on the ventilator. MV
significantly reduced (p < 0.05) maximal specific tension and the loss of force generating
ability fell over time (i.e., -18% after 12 hours and -46% after 24 hours of MV).
Our results of a 46% loss of specific tension are comparable to those of Le
Bourdelles et al. (92) who reported a 60% loss of specific tension after 48 hours of MV.
Further, our results indicate that the effect of MV is confined to the diaphragm as there
was no loss of soleus muscle mass. This observation is similar to the finding of Le
Bourdelles et al. (92) who reported no change in soleus or EDL maximal specific tension
after 48 hours of MV.
Mechanical Ventilation and Diaphragmatic Atrophy
After the time course experiments, we tested the hypotheses that short-term MV
(18 hours) would induce atrophy in all four fiber types in the diaphragm, increase the rate
of diaphragmatic muscle protein degradation, and increase oxidative stress in the
After 18 hours of MV there was no loss of body or soleus mass. However, total
diaphragmatic mass was significantly reduced (-6.9%) and this was primarily due to the
significant loss of costal diaphragmatic mass (-7.3%) (145). The atrophic effect of 18
hours of MV was confirmed using immunohistological techniques: the CSA of the type I
fibers was reduced -15%, IIa -27%, IId/x -30%, and IIb -24% (145). Again, these
findings support the postulate that MV results in diaphragmatic atrophy.
Measures of total and myofibrillar protein content were made to determine if the
observed contractile dysfunction and decreased diaphragmatic mass could be explained
by alterations in the protein composition of the diaphragm after MV. Eighteen hours of
MV resulted in significant reductions in diaphragmatic protein. Specifically, the
concentration of both myofibrillar protein and soluble protein significantly decreased by
-10%, resulting in a significant decrease in the total protein concentration (145).
Consistent with the loss of diaphragmatic mass, was the reduction in total (-16%) and
myofibrillar protein content (-16%), reflecting an absolute loss of protein from the
diaphragm (145). Additionally, MV resulted in a mean increase (-4%) in muscle water
The rate of protein degradation was also measured in vitro (145). After 18 hours of
MV two strips from the costal diaphragm were removed and suspended in separate in
vitro tissue chambers filled with a modified Krebs solution and aerated with 95% 02/ 5%
CO2 for 2 hours. The tyrosine concentration in the bathing medium was subsequently
analyzed fluorometrically (172). The rate of tyrosine release was used to determine the
rate of total protein catabolism because this amino acid is neither synthesized nor
degraded by skeletal muscle (164). The significant loss of protein, was due in part to the
significant increase in protein degradation, as indicated by a 28% increase in tyrosine
Our results also revealed that MV results in an increase in diaphragmatic oxidative
stress (145). The diaphragmatic content of both total 8-isoprostane and protein carbonyls
increased 30% and 35% respectively (145). Tissue levels of total 8-isoprostane and
protein carbonyls were measured as markers of lipid peroxidation and protein oxidation,
respectively. In the context of MV-induced diaphragmatic atrophy, an increase in protein
oxidation could be important because moderately oxidized proteins are more sensitive to
proteolytic attack by proteases (37, 38, 40, 96, 97, 118). Therefore, oxidative
modification of proteins could contribute to the elevated protein degradation measured
after MV. Further, the ubiquitin-proteosome pathway is the proteolytic pathway
implicated in the degradation of actin and myosin in muscle (55, 158) and this pathway is
up-regulated during periods of oxidative stress (142, 146, 161). An oxidative stress-
mediated up-regulation of the ubiquitin-proteosome pathway would lead to an increase in
protein degradation and thus atrophy.
Summary of Literature Review
MV is used to sustain patients with a life threatening illnesses, provide ventilation
during respiratory muscle failure ("respiratory muscle rest") or to compensate for a
damaged upper airway or to aid during recovery or rehabilitation from an illness.
Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary resection
can lead to respiratory muscle failure and require MV to maintain the life of the patient.
There are many modes of MV but controlled MV (CMV) results in complete
diaphragmatic inactivity and may be an ideal model to study the effects of MV. While
CMV provides the maximum degree of respiratory muscle rest it is not without
consequence to the very muscle it is intended to aid, the diaphragm. The reduced use of
the diaphragm while receiving MV may lead to atrophy and increase the likelihood of
diaphragmatic weakness. If the weakness is substantial it may be difficult to wean the
patient. The process of weaning is challenging and contributes to a large portion of the
The diaphragm is the primary muscle of inspiration and is chronically active
throughout life. The fiber type composition and the metabolic properties of the
diaphragm reflect the demand of this chronically active tissue. Studying the diaphragm is
often difficult to do using humans but the rat diaphragm has characteristics similar to the
human diaphragm, thus enabling difficult research questions to be addressed using this
The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly
adapts to its demand. Skeletal muscle hypertrophies when muscular activity increases,
whereas inactivity or disuse leads atrophy. Hypertrophy and atrophy, are therefore,
critically dependent on the relative rates of protein synthesis and protein degradation.
Atrophy results in a decrease in CSA and is functionally significant because muscle
strength is directly related to CSA.
A loss or decrease in neural stimulation of a muscle results in atrophy. There are
various models used in the study of hindlimb muscle atrophy (e.g., HI, spaceflight and
HU, and ST). Postural locomotor muscles rapidly adapt to the reduced load. Markers of
this adaptation include a loss of mass and CSA, slow-to-fast shift in MHC, and a
metabolic shift toward a more glycolytic fiber. The functional significance of these
adaptations is a loss of strength and endurance.
Manipulation of the phrenic nerve via denervation (phrenicectomy), tetrodotoxin,
or spinal cord isolation initially results in diaphragmatic hypertrophy followed by
atrophy. Paralyzing the diaphragm results in a significant decrease in specific tension
during all stages of its adaptation to paralysis. Attenuation of the contractile and
biochemical changes when the inactivity of the muscle fibers is matched to the inactivity
of the phrenic motoneurons, spinal cord isolation model, is an intriguing finding.
MV, in particular controlled MV, eliminates diaphragmatic EMG activity. The loss
of diaphragmatic electrical activity leads to a decrease in force generation and impairs
endurance. Specifically, MV leads to a loss of both in vivo and in vitro force generating
ability. The impairment in force generation is exacerbated by time spent on the ventilator.
The significant loss of diaphragmatic mass (i.e., atrophy) occurs in as few as 18 hours.
The loss of diaphragmatic mass includes the loss of both total and myofibrillar protein
and this is due in part to an increase in proteolysis.
The methods section is organized according to the objectives of each specific aim,
animals and experimental design, and the methods used.
Experimental Design-Specific Aim #1
The effect of MV on protein synthesis was investigated by testing the hypothesis
that MV-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of total
(mixed muscle protein (MMP)) and myosin heavy chain (MHC) protein synthesis.
Preliminary experiments indicated that prolonged MV results in significant
diaphragmatic atrophy. Both MMP and MHC protein synthesis rates (in vivo) in the
diaphragms of control, spontaneously breathing, and MV animals during 0 to 6, 6 to 12,
and 12 to 18 hours of MV were measured to determine the time course of changes in
Animals and Experimental Design
To address this specific aim, healthy young adult (4-month-old) female specific-
pathogen-free (SPF) Sprague-Dawley rats were individually housed and fed rat chow and
water ad libitum while being maintained on a 12 hour light/dark cycle for 3 weeks before
initiation of these experiments. Animals were randomly assigned to either the control or
MV experimental group. The control group was subdivided into four groups, control, or 6
to 18 hours of spontaneously breathing (SB 6, SB 12, SB 18). The control animals (n =
10) were not mechanically ventilated nor did they receive infusion of [1-13C]leucine. This
group was used to determine the natural abundance of [1-13C]leucine in the Sprague-
Dawley rat diaphragm. The SB 6 group (n = 10) was not mechanically ventilated but they
were infused with [1-13C]leucine while spontaneously breathing and anesthetized for 6
hours. Animals assigned to the SB 12 group (n = 10) were anesthetized for a total of 12
hours and [1-13C]leucine was infused during hours 6 tol2. Similarly, the SB 18 group (n
= 10) was anesthetized for a total of 18 hours and [1-13C]leucine was infused during
hours 12 to 18. The SB groups served as time matched controls for the MV groups.
The MV group was subdivided into three groups, MV 6, MV 12, and MV 18. The
MV groups were mechanically ventilated and the rate of protein synthesis was
determined after 6 (n = 10), 12 (n = 10), and 18 (n = 10) hours ofMV. [1-13C]leucine was
infused during the last 6 hours of the experimental period (i.e., 0 to 6, 6 to 12, and 12 to
Control (n=10) SB 6 (n=10) SB 12 (n=10) SB 18 (n=10) MV6(n=10) MV 12 (n=10) MV 18 (n=10)
no MV (acute) no MV no MV no MV
no cl3Leu cl3Leu infusion cl3 Leu infusion cl3 Leu infusion cl3Leu infusion cl3Leu infusion cl3Leu infusion
during hrs 0-6 during hrs 6-12 during hrs 12-18 during hrs 0-6 during hrs 6-12 during hrs 12-18
Figure 3-1. Experimental design for Specific Aim #1.
Mechanical ventilation protocol
Thirty minutes before anesthesia, animals received glycopryyloate (0.04 mg/kg IM)
in order to reduce airway secretions. Glycopryyolate was then administered every 2 hours
(0.04 mg/kg IM) for the remainder of the experiment. The animals were anesthetized
with an intraperitoneal (IP) injection of sodium pentobarbital (50 mg/kg of body weight).
Sodium pentobarbital was used as the general anesthetic because Le Bourdelles et al. (92)
have shown that the level of barbiturate required to maintain general anesthesia in rats
does not alter locomotor muscle contractile or biochemical properties. Additionally,
prolonged use of sodium pentobarbital (up to 18 hours) in rats does not induce atrophy in
locomotor muscles (i.e., soleus) (145). All surgical procedures were performed under
aseptic conditions. Once anesthetized, MV animals were tracheostomized and
mechanically ventilated with a volume-cycled ventilator (Inspira, Harvard Apparatus,
Cambridge, MA). A major advantage of volume-cycled ventilators is that tidal volume
remains relatively constant despite possible pathophysiological changes (e.g., airway
obstruction due to mucus secretion). Further, volume-cycled ventilators are capable of
maintaining a constant inspired percentage of oxygen (FIO2) which is important in
maintaining blood gas homeostasis during MV (20, 72, 165).
Heart rate and electrical activity of the heart was monitored via a lead II ECG using
needle electrodes placed subcutaneously. An arterial catheter was placed in the carotid
artery for constant measurement of blood pressure and blood samples (1 mL) were drawn
for analysis of [1-13C]leucine. Finally, a venous catheter was placed in the jugular vein to
add fluids and sodium pentobarbital.
Anesthesia was maintained during MV by intravenous infusion of sodium
pentobarbital (10 mg/kg body weight). Body (rectal) temperature was maintained at 370C
+10C by use of a computer controlled re-circulating heating blanket. Throughout MV
body fluid homeostasis was maintained via the administration of an intravenous
electrolyte solution, 2 mL/kg/hour. Continuing care during MV included expressing the
bladder, removal of airway mucus, lubricating the eyes, rotating the animal, and passive
movement of the limbs. Animals were continuously monitored during MV and while
spontaneously breathing, see below.
A board-certified pathologist (Dr. Sunjoo Kim, M.D.) performed all postmortem
procedures. Dr. Kim has extensive necropsy experience and is experienced in the clinical
microbiology techniques used to assess pneumonia and septicemia.
Necropsy examination. Necropsy examination included a detailed visual inspection
of the respiratory tract, the lungs, and the peritoneal cavity. Visualization of an abscess or
pus was considered a marker of infection, however no animals were infected.
Blood culture. Blood culture procedures were preformed according to the methods
recommended by the American Society for Microbiology (21) and Bailey and Scott's
Diagnostic Microbiology (82). A blood sample (0.5 mL) was drawn from each animal at
the conclusion of the experimental period via the jugular vein. Each sample was
immediately inoculated directly into the MacConkey agar plate (82). The blood cultures
were incubated at 370C for 5 days and inspected daily (82). Observation of bacterial
growth on the culture media would have been considered evidence of sepsis.
Control animals (nonmechanically ventilated) protocol
The animals in the control groups were randomly placed into one of four groups:
Control (acute anesthesia, no MV, no [1-13C]leucine infusion) or SB 6, SB 12, or SB 18
(anesthetized, spontaneously breathing, [1-13C]leucine infusion). Control animals (acute
anesthesia) were free of intervention during the hours before removal of the diaphragm
for measurement of biochemical properties. That is, these animals were not mechanically
ventilated before study. Control animals received an IP injection of sodium pentobarbital
(50 mg/kg body weight); after a surgical plane of anesthesia was reached their
diaphragms were removed for subsequent measurements of biochemical properties. SB 6,
SB 12, and SB 18 animals received the same surgical intervention and [1-13C]leucine
infusion paradigm as the MV animals except these animals were not mechanically
ventilated (i.e., they were breathing on their own during the entire experimental period).
The MV and SB animals received the same continuing care during the experimental
period and post mortem examination after the experimental period.
Methods Used: Biochemical Assays
Tissue removal and storage
At the appropriate times (as specified in the experimental design) biochemical
studies were conducted on muscle samples taken from the costal portion of the
diaphragm. Costal diaphragm segments obtained for biochemical analysis were rapidly
frozen in liquid nitrogen and stored at -800C until assay.
Rates of in vivo diaphragmatic protein synthesis
Infusion. The fractional rate of diaphragm muscle protein synthesis was measured
using intravenous infusion of [1-13C]leucine (Cambridge Isotopes Laboratory, Andover,
MA) and quantifying the in vivo rate of incorporation of [1-13C]leucine into both total and
contractile proteins in the diaphragm by gas chromatography-combustion-isotope ratio
mass spectrometry (GC-C-IRMS). The stable isotope [1-13C]leucine was chosen for
* Less isotope effect (i.e., less tissue injury).
* Small tissue sample is required for analysis.
* Validity and reliability of this label for measurement of the rate of in vivo muscle
protein synthesis is well established (121).
Animals were anesthetized with sodium pentobarbital and a tygon catheter was
placed in the jugular vein for infusion of [1-13C]leucine. A polyethylene catheter was also
placed in the carotid artery for blood sampling. Note, the jugular and carotid catheters
were the same as described above for adding electrolyte solution and sodium
pentobarbital and for monitoring arterial blood pressure. The jugular vein and carotid
artery were catheterized in the SB and MV groups immediately after anesthetization. The
catheter was kept patent by periodically flushing with heparinized saline (20 U/mL). At
the beginning of the infusion, animals were primed with 1.6 mg [1-13C]leucine/100 grams
of body weight, followed by a constant infusion rate of 0.20 mg[l-13C]leucine/100 grams
of body weight/ hour for 6 hours (KD Scientific Model 100 syringe pump, Boston, MA).
This infusion rate was chosen on the basis of previous experiments indicating that this
rate results in optimal muscle [1-13C]leucine enrichment for the determination of protein
synthesis in rat skeletal muscle (181). The infusion of the labeled amino acid is delivered
such that -5-10% of the plasma pool of free amino acids is labeled (130). In this sense
the labeled amino acid acts as a tracer with little or no effect on the overall metabolism of
tissue being investigated (130). The duration of the infusion period need only be long
enough for enrichment of the target protein but not so long as to allow recycling of the
tracer (130). Skeletal muscle protein turns over slowly and thus tracer recycling does not
present a problem (130).
Immediately before infusion, and at the end of the 5th and 6th hours of infusion,
blood samples (1 mL) were drawn for the measurement of plasma a-ketoisocaproate (a-
KIC) enrichment. At the completion of the experimental period, the costal diaphragm was
rapidly removed, frozen in liquid nitrogen, and stored at -800C until assay. Note that the
infusion pump continued to infuse at the time of tissue removal. A tissue sample only
needs to be taken at the end of the infusion period if no other labeled amino acid has been
administered previously. The baseline sample labeling can be assumed to be close to
previously measured tissue from the same tissue population (57) or from mixed blood
Sample analysis and calculations. Plasma a-KIC was isolated, prepared as the
trimethylsilyl quinoxalinol derivative, and analyzed for [1-13C]leucine abundance by use
of gas chromatography-electron impact quadrupole-mass spectrometry (GC-MS) (HP
5890 Series II and GC/HP 5970 Series MS, Hewlett-Packard, Avondale, PA). Tissue
fluid-free amino acids were extracted by homogenization in 10% TCA. The N-
heptafluorobutyryl propyl esters were formed, and the [1-13C]leucine abundance was
determined using electron capture-negative chemical ionization GC/MS (HP 5988A
Series II and GC/HP 5970 Series MS, Hewlett-Packard). The plasma a-KIC enrichment
and the tissue fluid [1-13C]leucine enrichment [in mole percent in excess (MPE)] were
used to represent the precursor pool (leucyl tRNA) enrichment for the calculation of
protein synthesis rate (Ks; the percentage of the protein mass synthesized per hour
(%/hour)). The following equation was used to calculate Ks
[1 -13 C] leucine MPE enrichment in protein x 100
K s =---------------
precursor pool enrichemnt x (t, t )
where (t2-tl) is the infusion time (hours).
The rate of protein synthesis can be determined if the site of the incorporation of
the amino acid into protein (i.e., in the appropriate aminoacyl-tRNA pool, is known)
(130). However since identification of the correct pool presents a problem (130), a
surrogate index of the labeling of the aminoacyl-tRNA pool has been adopted. The
branched chain amino acid leucine is the preferred tracer and the labeling of its
transamination product, a-ketoisocaproate (c-KIC), is measured. The use of a-KIC as
the surrogate of the true precursor labeling is possible because the formation of its
transamination product, a-KIC, occurs intracellularly and thus a-KIC reflects the extent
of labeling in the true precursor pools for protein synthesis (130). Indeed, the labeling of
a-KIC after infusion of labeled leucine is within 10% of the labeling of tRNA in human
skeletal muscle (174).
Analysis of mixed muscle protein synthesis rates. To determine the [1-13C]leucine
abundance in mixed muscle protein (MMP) 50-60 mg muscle samples were homogenized
in 1 mL of 10% TCA and hydrolyzed in 6 N HC1 at 1100C for 24 hours. The n-acetyl n-
propyl (NAP) esters of the component amino acids were formed, and the [1-13C]leucine
abundance in the hydrolyzed MMP were determined using GC-C-IRMS according
Yarasheski et al. (181).
Isolation of MHC for analysis of synthesis rates. All procedures for the MHC
extractions were performed on ice or at 40C. Frozen muscle samples (60-80 mg) were
homogenized in 1 mL of a 250 mM sucrose buffer (in mM: 250 sucrose, 100 KC1, 5
EDTA, and 20 imidazole, pH 6.8). The homogenate was centrifuged at 1,200 x g for 10
minutes, and the supernatant was discarded. The pellet was suspended in 1 mL of a 0.5%
Triton X-100 solution (175 mM KC1, 0.5% Triton X-100, pH 6.8), a modification of
Solaro et al. (150). The suspension was homogenized and centrifuged as before. This step
removes many of the soluble matrix proteins. The resultant pellet was rinsed (i.e.,
homogenized and centrifuged) with 1 mL wash buffer (150 mM KC1 and 20 mM Tris, pH
7.0) to remove excess Triton X-100 solution. The pellet was then frozen at -800C. The
pellet was resuspended in SDS buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.001%
bromophenol blue, and 5% P-mercaptoethanol) and boiled for 5 minutes.
These extracts of myofibrillar protein (containing ~1 mg) were separated by SDS-
PAGE. All electrophoresis chemicals were purchased from Bio-Rad (Hecules, CA). Each
extract was separated on an individual gel with a single wide lane, utilizing a 7% T-2.5%
C polyacrylamide slab gel with a 4% T-2.5% C stacking gel. The proteins were separated
(150 volts, -5 hours) using a discontinuous buffer system that useed a Tris-Tricine buffer
(1.6 mM Tris, 16 mM Tricine, 0.01% SDS, pH 6.4) and a Tris buffer (2.5 mM Tris,
0.01% SDS, pH 6.4) as the cathode and anode buffers, respectively.
The separated protein was visualized by Coomassie staining (0.1% Coomassie
brilliant blue R-250, 45% methanol, and 10% glacial acetic acid) for 10 to 15 minutes.
after overnight destaining (30% methanol and 10% glacial acetic acid), the band
corresponding to the molecular mass of MHC was identified. The protein band was
carefully cut out within the distinctly stained boundary, minced, and put into a tube. The
samples were hydrolyzed in 3 to 4 mL of concentrated HC1 (110C for 48 hours), and the
NAP esters of the amino acids were prepared for analysis of [1-13C]leucine abundance
Measuring mixed muscle (MMP) and MHC protein synthesis is very involved but
the method is well established and in everyday use in the Yarasheski laboratory. The
method of accurate identification of the MHC band, the quantity and quality of the band,
and the reliability of the isotopic enrichments are well documented (70).
Planned comparisons were made between relevant groups with a Bonferroni
correction for the number of comparisons conducted. Significance was established at
p < 0.05.
Experimental Design-Specific Aim #2
The effect of MV on MHC mRNA content was determined by testing the
hypothesis that MV alters pretranslational events in the diaphragm. Preliminary
experiments indicated that prolonged MV results in significant diaphragmatic atrophy
and the proposed experiments determined the time course of changes in MHC mRNA
during 6 to 18 hours of MV. To test our hypothesis we measured type I, IIa, IId/x, and IIb
MHC mRNA content in diaphragms from control, SB, and MV animals after 6, 12, and
18 hours of SB or MV. The measurements were made using a portion of the costal
diaphragm from the same group of animals used to test the first hypothesis. The
experimental design, surgical procedures, care, diaphragm removal, necropsy
examination, and blood culture have been described in Specific Aim #1.
Methods Used: Biochemical Assays
Total RNA isolation
A portion of the costal diaphragm, -50 mg, was homogenized in 1.5 mL of Trizol
(Invitrogen, Carlsbad, CA) and processed according to the manufacture's instructions.
This protocol is based on the method described by Chomczynski and Sacchi (34). The
sample was centrifuged at 12,000 x g for 10 minutes to the remove insoluble material.
The RNA portion, the upper aqueous phase, was transferred and incubated at room
temperature for 5 minutes. The RNA was extracted with bromochloropropane,
precipitated with isopropanol, washed with 75% ethanol, and pelleted via centrifugation.
The pellet was resuspended in RNAse free water (Sigma, St. Louis, MO). The
concentration and purity of the total RNA extracted was measured spectrophotometrically
at 260 nm and at 280 nm in lx TE buffer (Promega, Madison, WI). Ideally, the ratio of
A260/A280 should be greater than 1.8. This is a measure of RNA purity. Absorbance at 260
nm (A260) reflects the RNA contration and absorbance at 280 nm (A280) reflects the
protein content. The concentration of RNA was determined via the equation:
OD at 260 x 40 1
ugRNA IpuL = x-
# of /L read 10
The optical density (OD) of RNA at 260 nm (OD260) is assumed to equal 40 [tg/[tL (178).
This method yields un-degraded RNA, free of DNA and proteins.
The integrity of the extracted total RNA was verified by gel electrophoresis of 1 |tg
RNA on an 1% agarose tri(hydroxymethyl)aminomethane (Tris)-borate-EDTA buffer
(TBE) gel containing ethidium bromide using lx TBE as the running buffer. Both the
28S and 18S ribosomall RNA) bands were clear and distinct in the intact samples.
Samples that did not demonstrate these characteristics (i.e., degraded samples) were
discarded and another tissue sample from the same animal was processed and an un-
degraded RNA sample was then analyzed. The total RNA samples were then stored at -
800C until analysis.
Reverse transcription (RT)
Total RNA was reverse transcribed and amplified via PCR for each diaphragm
sample. Briefly, 1 |tg of total RNA was reverse transcribed using SuperScript II RT
(Invitrogen, Carlsbad, CA) and a mix of oligo dT (100 ng/reaction) and random primers
(200 ng/reaction) in a final volume of 20 ptL according to the protocol provided by the
manufacturer. An equal number of samples from each group were included in each run.
The samples were then stored at -800C until used for PCR.
Polymerase chain reaction (PCR)
MHC content was determined via relative RT-PCR with 18S serving as the internal
standard (Ambion, Austin TX). The MHC primer sequences used have been published by
Dr. Ken Baldwin's group (178) and are shown in Table 3-1. The primer for the five prime
(5') end of each mRNA was designed from a highly conserved region in all known rat
MHC genes -600 base pairs upstream of the stop codon (99). The four adult rat MHC
isoforms (I, IIa, IId/x, and IIb) are identical in this region. This allowed for the design of
a "common" primer with the following sequence:5' GAA GGC CAA GAA GGC CAT C
3' (178). The primers for the three prime (3') end were derived from the 3'untranslated
regions (UTR) of each of the different MHC genes, the sequences for each rat MHC gene
are highly specific in this region (42, 65, 66).
In order to account for differences in the initial amount of total RNA and to serve
as an internal standard, 18S ribosomal RNA was co-amplified with the target cDNA
(mRNA) in each PCR sample. The Alternate 18S Internal Standard kit (Ambion, Austin,
TX) was used. The 18S primers were mixed with the provided competimer in a 1:4 ratio.
The 18S competimer is required to decrease the 18S signal. The 18S primer to
competimer ratio was optimized such that amplification of the target cDNA and 18S
ribosomal RNA was similar (Ambion, Relative RT-PCR kit protocol).
The PCR conditions were as follows: 2 mM MgC12 in standard PCR buffer
(Invitrogen, Carlsbad, CA), 0.2 mM dNTP, 0.2 [tM of the common primer, 0.2 [tM of
one of the four gene specific primers, 0.5 [tM 18S primer/competimer mix, 2 p.L of the
diluted RT product (1 pIL of each RT reaction was diluted 40 fold before PCR
amplification), and 0.75 units of Taq polymerase (Invitrogen, Carlsbad, CA) in a final
volume of 25 ILL. All four MHC genes were amplified in four separate reactions for each
experimental group and one sample form each experimental group was included in each
PCR run. PCR was carried out with an initial 3 minute denaturation step at 960C,
followed by 24 cycles, each cycle consisting of 45 seconds at 960C (denaturation), 60
seconds at 500C (primer annealing), 90 seconds at 720C (extension), and a final step of 3
minutes at 720C using the Stratagene Robocycler. The number of cycles was determined
to be on the linear portion of a semilog plot of the yield (see below) expressed as a
function of cycle number. The PCR products were separated by agarose gel
electrophoresis [20 pIL sample of the PCR product loaded on 2.0% agarose gels (in lx
TBE buffer) containing 0.2 [tg/mL ethidium bromide] for visualization of the PCR
Analysis of gels
A digital image of each gel was captured and the bands were analyzed via
computerized densitometry (Gel-Doc 2000, Bio-Rad, Hercules, CA). The integrated areas
of each target (MHC) and 18S internal control fragment DNA band were determined with
the local background subtracted. The integrated area of the target band was normalized to
the integrated area of the corresponding 18S internal control fragment, thus correcting for
any differences in PCR reaction efficiencies. The value for each MHC mRNA is
expressed as MHC mRNA/18S.
Planned Comparisons were made between relevant groups with a Bonferroni
correction for the number of comparisons conducted. Significance was established at
p < 0.05.
Table 3-1. Oligonucleotide primers used for the PCR reactions
MRNA Common Primer Antisense Primer Sample
(5' end) (3' end) cDNA
Type I MHC
Type IIa MHC
Type IId/x MHC
Type IIb MHC
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GGT CTC AGG
GCT TCA CAG GC 3'
5' TCT ACA GCA
TCA GAG CTG CC 3'
5' GGT CAC TTT
CCT GCT TTG GA 3'
5' GTG TGA TTT
CTT CTG TCA CC 3'
Sample cDNA is the size of the myosin heavy chain (MHC) mRNA PCR product in base
common stop codon
region -600 bp MHC sequences 3'UTR
Figure 3-2. Schematic representation of the myosin heavy chain (MHC) genes, the
common primer is identical in all sequences, followed by -600 base pairs (bp)
of coding sequence. A stop codon and 3'-untranslated region (UTR) that are
highly specific for each MHC gene with little or no sequence similarity
among gene family members are depicted.
Morphological, Physiological, and Post Mortem Observations
The body mass characteristics of each experimental group are presented in Table 4-
1. No significant differences (p > 0.05) existed in pre-experiment or post-experiment
body mass between groups. Importantly, no group experienced a significant (p > 0.05)
loss of body mass over the course of the experiment, indicating adequate hydration
during the experimental period. Additionally, all animals urinated and experienced
intestinal transit during the experimental period.
Heart rate and systolic blood pressure were monitored as a means of determining
animal homeostasis during the experimental period. The mean heart rate and systolic
blood pressure data are presented in Tables 4-2 and 4-3, respectively. Heart rate and
blood pressure were within normal ranges and were well maintained during the
experiments. Further, note that the heart rate and blood pressure responses were very
similar between the SB animals and the MV animals at each time point.
Post mortem examination of the SB and MV animals included a necropsy and
blood culture. No animals demonstrated any signs of infection, weight loss, or post
mortem abnormalities, and all blood cultures were negative for bacteria. Additionally, the
colonic temperature of each animal remained constant, 37 + 0.5C, during the
experiments. Collectively, these results indicate that our aseptic surgical technique
successfully prevented infection.
Influence of Mechanical Ventilation on Protein Synthesis
MV resulted in depressed protein synthesis in the diaphragm within the first 6
hours; this reduced rate of protein synthesis persisted throughout the remainder of the
experimental period. Note that within the first 6 hours, the rate of both mixed muscle
protein (MMP) (-30%) and MHC (-65%) protein synthesis significantly decreased
(p < 0.05). Precursor pool 13C enrichment, MMP and MHC 13C enrichment, and MMP
and MHC fractional synthetic rates are each presented in Figures 4-1, 4-2 and 4-3.
Enrichment values are expressed as mole percent in excess (MPE). Mole percent in
excess is the enrichment of 13C above natural levels. Plasma enrichment of 13C in MPE
was determined from the baseline plasma sample in each animal before infusion. Tissue
fluid, MMP, and MHC 13C enrichment in MPE was calculated using the acute anesthesia
animals as the baseline measure of naturally occurring levels of 13C.
Figure 4-1 reports plasma [13C]leucine and [1-13C]ketoisocaproic acid ([13C]KIC)
enrichment. The plateau in the enrichment of the plasma precursor pools indicates that a
steady-state was achieved by the 6th hour of infusion. No significant differences
(p > 0.05) existed in the enrichment of plasma [13C]leucine or [13C]KIC after 5 hours of
infusion compared to 6 hours of infusion within groups (e.g., SB 6 at hour 5 vs. hour 6).
Additionally, the enrichment of plasma [13C]leucine or [13C]KIC did not differ between
groups (e.g., SB 6 vs. MV 6). Rates of protein synthesis calculated using the plasma
[13C]leucine and [13C]KIC precursor pools were made using the 6 hour enrichment
The [13C]leucine enrichment of the tissue fluid precursor pool did not differ
between time matched groups (Figure 4-2). Note that the 18 hour SB and MV groups
each experienced significantly greater (p < 0.05) tissue fluid [13C]leucine enrichment than
did their 6 and 12 hour counterparts.
By averaging the endogenous amount of 13C in the diaphragms from the Control
acute anesthesia group and subtracting the amount of endogenous 13C from all SB and
MV values we derived the degree of 13C enrichment of the diaphragm proteins. The
difference between the endogenous 13C content and the measured 13C after [13C]leucine
infusion is termed the degree of enrichment.
The SB group experienced a significant (20%, p < 0.05) decrease in diaphragmatic
MMP [13C]leucine enrichment over time (hour 6 to hourl8). In comparison to their time
matched counterparts, the MV animals experienced a significant (30-34%, p < 0.05)
decrease in diaphragmatic MMP [13C]leucine enrichment (Figure 4-3). [13C]leucine
enrichment of MHC protein in the diaphragm remained constant over time in the SB
group. A significant (68 to 75%, p < 0.05) decrease in the enrichment of diaphragm MHC
protein was observed during MV at each time point (Figure 4-3).
After measurement of precursor pool (plasma and tissue fluid) and MMP and MHC
enrichment, calculation of the fractional synthetic rate of protein synthesis was
performed. The fractional synthetic rate of both MMP and MHC protein was calculated
using all three surrogates of [13C]leucy-tRNA: plasma [13C]leucine, plasma [13C]KIC, and
tissue fluid [13C]leucine. MV significantly (p < 0.05) slowed the fractional synthetic rate
of both MMP and MHC protein synthesis (Figure 4-4, Figure 4-5, and Table 4-4).
Regardless of which precursor pool was used for these calculations the decreased
fractional synthetic rate remained. However, for clarity, only the fractional synthetic rate
calculated using the tissue fluid [13C]leucine precursor pool is presented in graphic form
(Figure 4-4 and Figure 4-5).
Mixed muscle protein synthesis, a measure of whole muscle protein synthesis,
slowed significantly (30%, p < 0.05) during the first 6 hours of MV as compared to the
time matched SB 6 group (Figure 4-4 and Table 4-4). The 30% decrease in the rate of
MMP synthesis persisted at hour 12 (MV 12 compared to SB 12, -26%) and hour 18 (MV
18 compared to SB 18, -29%).
Myosin heavy chain protein synthesis was measured in order to estimate the impact
of MV of the rate of contractile protein synthesis. Within the first 6 hours of MV, MHC
protein synthesis slowed significantly (66%, p < 0.05) as compared to the time matched
SB 6 group (Figure 4-5 and Table 4-4). In parallel with the MMP synthesis rates, the
decrease in MHC protein synthesis rates after MV remained constant as compared to
each time matched SB group.
Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous
Breathing and Mechanical Ventilation
Total RNA is -80-85% ribosomal RNA (rRNA) and can be used as an index of the
quantity of ribosomal subunits and as an indirect index of the synthetic capacity of the
tissue. In contrast, mRNA constitutes -2-3% of the total RNA pool. Total RNA was
isolated from each diaphragm and the mRNA encoding the four adult MHC phenotypes
was then measured to determine if the observed decrease in protein synthesis was due, in
part, to a decrease in total RNA and/or MHC mRNA. The total RNA findings are
presented in Table 4-5. Exposure to the anesthetic (SB groups) or MV did not affect the
amount of total RNA isolated from the diaphragms of any of the groups.
Once isolated from the diaphragm, total RNA was reverse transcribed. The cDNA
and the 18S rRNA control fragment were then amplified via PCR and the products were
separated electrophoretically on 2% agarose gels stained with ethidium bromide. An
example of the products is depicted in Figure 4-6. In each lane (top to bottom) the two
products, the amplified target mRNA and the amplified 18S internal standard fragment,
were present. The upper band is the amplified target mRNA and the lower band is the
18S rRNA internal standard fragment. The expected size of each amplified target mRNA
product is 596 bp, 570 bp, 574 bp, 590 bp for type I, IIa, IIx, and lib MHC, respectively.
The expected 18S control fragment size is 324 bp.
Figure 4-7 through Figure 4-10 shows MHC mRNA results. The MHC mRNA data
are expressed relative to the 18S rRNA internal standard product to account for variations
in the RT-PCR process. Exposure to prolonged anesthesia (SB) or MV did not alter the
relative amount of any of the four MHC mRNA's. The graphic results for each MHC
mRNA are depicted in Figure 4-7 through Figure 4-10.
Table 4-1. Animal body mass before and after experimental period
Group Body Mass (g) before Body Mass (g) after
Control 255.0 + 6.8 ----
SB 6 251.9 3.6 251.7 3.6
MV 6 248.5 + 4.2 248.5 4.2
SB 12 259.7 + 3.0 258.3 + 3.0
MV 12 262.8 + 2.5 262.7 2.3
SB 18 259.0 + 3.8 259.4 4.2
MV 18 259.7 + 3.1 259.4 + 3.3
Values are means standard error (SE) with n = 10 per group. SB = spontaneously
breathing. MV = mechanically ventilated.
Table 4-2. Heart rate response during MV and SB
Group Time zero 6th hour 12th hour 18th hour
SB 6 343 9 352 6
MV 6 357 3 360 6
SB 12 351 5 361 1 350 10
MV 12 375 7 358 14 364 + 6
SB 18 340 12 360 11 324 10 322 22
MV 18 374 10 363 5 388 11 395 17
Values are means SE expressed in beats per minute with n = 10 per group. N/D = no
data. SB = spontaneously breathing. MV = mechanically ventilated.
Table 4-3. Systolic blood pressure response during MV and SB
Group Time zero 6th hour 12th hour 18th hour
SB 6 135 4 109 5
MV 6 122 3 108 5
SB 12 137 3 129 6 103 7
MV 12 154 6 107 11 106 8
SB 18 128 5 112 5 96 5 92 3
MV 18 138 3 110 5 100 8 98 7
Values are means SE expressed in mmHg with n = 10 per group. N/D = no data. SB =
spontaneously breathing. MV = mechanically ventilated.
-*- SB 6
-i- SB 12
-U- SB 18
Tracer Infusion Time (hr)
Plasma [13C]leucine and plasma [13C]ketoisocaproic acid ([13C]KIC)
enrichment. Values are means SE expressed in mole percent in excess of
natural 13C abundance with n = 10 per group. SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Hour 5 = plasma
[13C]leucine or [13C]KIC after 5 hours of infusion; hour 6 = plasma
[13C]leucine or [13C]KIC after 6 hours of infusion.
E e T *
SB 6 MV 6 SB 12 MV12 SB 18 MV18
Figure 4-2. Tissue fluid [13C]leucine enrichment in the diaphragm. Values are means +
SE expressed in mole percent in excess of natural 13C abundance measured in
Control animal diaphragm with n=10 per group. SB = spontaneously
breathing animals; MV = mechanically ventilated animals. $ Significantly
different (p < 0.05) from SB 18; significantly different (p < 0.05) from MV
: 1 HC
SB6 IW6 SB12 IW12 SB18 IW18
Figure 4-3. Mixed muscle protein and myosin heavy chain [13C]leucine enrichment in the
diaphragm. Values are means + SE expressed in mole percent in excess of
natural 13C abundance measured in Control animal diaphragm with n=10 per
group. MMP= mixed muscle protein; MHC = myosin heavy chain; SB =
spontaneously breathing animals; MV = mechanically ventilated animals. *
Significantly different (p < 0.05) from time matched SB; t significantly
different (p < 0.05) from SB 6, t significantly different (p < 0.05) from MV
SB 6 MV6 SB 12 MV12 SB 18 MV18
Figure 4-4. Fractional synthetic rates of mixed muscle protein (MMP) by calculation with
tissue fluid [13C]leucine as the surrogate measure of the [13C]leucyl-tRNA
precursor pool. Values are means + SE expressed in percent per hour (%/hr)
with n=10 per group. SB = spontaneously breathing animals; MV =
mechanically ventilated animals. Significantly different (p<0.05) from time
matched SB group; significantly different (p < 0.05) from SB6.
Fractional synthetic rates of myosin heavy chain (MHC) protein by
calculation with tissue fluid [13C]leucine as the surrogate measure of the
[13C]leucyl-tRNA precursor pool. Values are means + SE expressed in
percent per hour (%/hr) with n=10 per group. SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Significantly different
(p<0.05) from time matched SB group; significantly different (p < 0.05)
from SB 6.
S6 V6 S 12 V12 S18 V18
SB 6 MV 6 SB12 MV12 SB18 MV18
Table 4-4. Fractional synthetic rates of mixed muscle protein and myosin heavy chain
protein by calculation with each surrogate of the [13C]leucyl-tRNA precursor
6 hour SB
6 hour MV
12 hour SB
12 hour MV
18 hour SB
18 hour MV
Plasma [13C]leucine (%/hr)
0.1381 + 0.0077 0.0514 + 0.0068
0.0886 + 0.0077 0.0178 0.0056 *
0.1013 0.0044 8 0.0487 + 0.0088
0.0624 0.0066 t 0.0173 0.0104 *
0.1174 0.0055 8 0.0419 0.0056
0.0813 0.0055 0.0118 0.0027 *
Plasma [13C]KIC (%/hr)
6 hour SB
6 hour MV
12 hour SB
12 hour MV
18 hour SB
18 hour MV
0.2454 + 0.0127
0.1747 0.0110 *
0.1281 + 0.0110 t
0.2277 + 0.0110
0.0920 + 0.0132
0.0321 + 0.0088 *
0.0864 + 0.0121
0.0304 + 0.0176*
0.0774 + 0.0099
0.0225 + 0.0051*
Table 4-4. (continued)
Tissue Fluid [13C]leucine (%/hr)
Group MMP MHC
6 hour SB 0.2040 + 0.0110 0.0990 + 0.0180
6 hour MV 0.1691 0.0110 0.0341 + 0.0092*
12 hour SB 0.1879 0.0088 8 0.0899 + 0.0165
12 hour MV 0.1388 0.0088 t 0.0310 + 0.01650*
18 hour SB 0.1636 0.0077 0.0584 0.0048
18 hour MV 0.1164 0.0055 t 0.0187 0.0044*
Values are means SE expressed in percent per hour with n= 10 per group. MMP =
mixed muscle protein; MHC = myosin heavy chain; SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Significantly different (p<0.05) from
time matched SB group; significantly different (p < 0.05) from SB 6, t significantly
different (p < 0.05) from MV 6. Note that the Tissue Fluid [13C]Leucine data are also
presented in Figures 4-4 and 4-5.
Table 4-5. Total RNA obtained from the costal diaphragm
Group Total RNA ([tg/mg)
Control 0.923 0.200
6 hour SB 0.937 0.032
6 hour MV 0.962 0.022
12 hour SB 0.898 0.017
12 hour MV 0.910 + 0.018
18 hour SB 0.959 0.028
18 hour MV 0.991 + 0.023
Values are means SE expressed as |tg of total RNA and as the amount ([tg) of total
RNA per mg wet weight of diaphragm with n = 10 per group. SB = spontaneously
breathing; MV = mechanically ventilated.
Figure 4-6. RT-PCR products separated on a 2% agarose gel with n=10 per group. C =
Control, acute anesthesia animals; SB = spontaneously breathing animals;
MV = mechanically ventilated animals; MWM = molecular weight marker in
basepairs (bp); T = target MHC mRNA; 18S = 18S rRNA internal standard.
0 I I I I I I I
Control SB 6 MV6 SB 12 MV12 SB 18 MV18
Figure 4-7. Relative type I MHC expression. Values are means + SE expressed as type I
MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S)
with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.
" SB6 MV6 SB12 MV12 SB18 MV18 C SB6 MV6 B172 MV12 SB18 MV18
" SB MV6 SB12 MV12 ;ii18 MV18 C SB6 MV6' SB12 MV12 SB18 MV18
Control SB 6 MV 6 SB 12 MV 12 SB 18 MV 18
Figure 4-8. Relative type IIa MHC expression. Values are means + SE expressed as type
IIa MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.
Control SB 6 MV 6 SB 12 MV12 SB 18 MV 18
Figure 4-9. Relative type IIx MHC expression. Values are means + SE expressed as type
IIx MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.
Control SB 6 MV 6 SB 12 MV12 SB 18 MV18
Figure 4-10. Relative type IIb MHC expression. Values are means + SE expressed as type
lib MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.
Overview of Principle Findings
These experiments investigated the affect of 6 to 18 hours of MV on protein
synthesis and MHC mRNA in the rat diaphragm. Our results support the hypothesis that
the MV-induced diaphragmatic atrophy is, at least in part, due to a decreased rate of total
(MMP) and myosin heavy chain (MHC) protein synthesis. Indeed, within the first 6 hours
of MV MMP synthesis decreased by -30% and MHC protein synthesis decreased by
-65%. These decrements in protein synthesis persisted throughout the 18 hours of MV.
In contrast, our data do not support the postulate that MV alters pretranslational events in
the diaphragm as indicated by the observation that MV did not alter MHC mRNA
content. A detailed discussion of these points follows.
Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm
Mixed Muscle Protein Synthesis
MMP synthesis is the average synthetic rate of all proteins (e.g., contractile
proteins, sarcoplasmic reticulum proteins, enzymatic proteins) in the muscle sample and
was measured as an index of total protein anabolism in the diaphragm. The fractional rate
of MMP synthesis in the diaphragm was measured after three periods of MV (6, 12, and
18 hours), and compared to time matched controls. The SB 6 MMP synthetic rate
calculated using tissue fluid [13C]leucine was -0.2 %/h. These results are lower than
published MMP synthetic rates in the rat diaphragm, -0.4%/h to 0.6%/h (50, 127, 169). It
should be noted that young (-100g) rapidly growing animals were used in these previous
studies and this may have contributed to the differences in the rates. However, our
measured protein synthetic rates are similar to those measured in other adult rat skeletal
muscles (e.g., 0.16%/h in quadriceps (16), 0.23%/h in gastrocnemius (28), and 0.33%/h
in soleus (162)). Hence, the measured rates of MMP synthesis in the rat diaphragm in the
current study are consistent with rates reported in the literature for adult animals.
The MV-induced 30% decrease in MMP synthesis in the diaphragm occurred
during the first 6 hours of MV. Additionally, the MV-induced decrement in protein
synthesis remained steady after 12 and 18 hours of MV. This rapid change in skeletal
muscle protein synthesis has also been observed during periods of inactivity in rat
gastrocnemius. Indeed, 6 hours of hindlimb immobilization leads to a 37% decrease in
MMP synthesis (28). Similarly, the observed decrease in MMP synthesis in immobilized
rat gastrocnemius remains at this level after 2 days of immobilization (168). Therefore,
the observed decrease in MMP synthesis after 12 and 18 hours of MV in the rat
diaphragm is consistent with the rat hindlimb immobilization data (168) and indicates
that the diaphragm, like other skeletal muscles, is sensitive to loading state. Once
unloaded, via MV, protein synthesis in the diaphragm rapidly decreases and a new
steady-state of protein synthesis is established.
Myosin Heavy Chain Protein Synthesis
MHC is an essential component of the contractile apparatus and constitutes -25%
of skeletal muscle mass (122, 182). Importantly, force generation is proportional to the
amount of myofibrillar protein within the fiber and thus a decrease in the rate of MHC
synthesis would lead to a decrease in the force generating ability of the diaphragm (17,
26, 125). Accordingly, the rate of MHC protein synthesis was measured after 6, 12, and
18 hours of MV and compared to time matched controls.
These were the first experiments to measure the rate of MHC protein synthesis in
the rat diaphragm. The rate of protein synthesis calculated using tissue fluid [13C]leucine
is -0.1%/h in contracting diaphragm (SB 6). Previous investigations report MHC protein
synthesis rates of 0.1%/h in the rat quadriceps (16) and -0.25%/h in the rat soleus (162).
It is unclear why the rates in the diaphragm are not in line with those of the more active
The observation that MHC protein synthesis rate is slower than the MMP synthesis
rate suggests that MHC protein turns over slower (longer half-life) than other proteins in
the MMP pool. The rates of MHC protein synthesis in the current study are -60% less
than the MMP synthesis rate. Previous studies report an -40% difference in quadriceps
(16) and an -25% difference in soleus (162). The data in the current study suggest that
the MHC protein pool in the diaphragm is turning over slower than MHC protein in other
Six hours of MV resulted in an -65% decrease in the rate of MHC protein
synthesis. This decrease in MHC protein synthesis was maintained through 18 hours of
MV. This rapid decrease in the rate of diaphragmatic MHC protein synthesis was more
severe than the reported change after 5 hours of hindlimb unloading where a consistent
but non-significant decrease was measured in the soleus (162). A possible explanation for
the divergent findings is that during MV the diaphragm is not contracting but the soleus
is free to contract (against little resistance) during hindlimb unloading. Hence, this level
of activation in the soleus may serve to attenuate the decrease in protein synthesis during
hindlimb unloading compared to MV. The change in MHC protein synthesis has not been
measured during hindlimb immobilization but the synthetic rate of another essential
contractile protein, a-actin, has been examined (173). During the first 6 hours of
hindlimb immobilization, a-actin protein synthesis in the rat gastrocnemius decreases
-66% (173). The MHC protein synthesis data in the current study and the a-actin protein
synthesis data after 6 hours of hindlimb immobilization (173) indicate that skeletal
muscle rapidly adapts to unloading by significantly decreasing the rate of contractile
Myosin Heavy Chain mRNA
The fractional synthetic rate of specific proteins can be altered by pretranslational
events leading to a decrease in the amount of a given mRNA (e.g., the rate of
transcription or turnover of MHC mRNA). As discussed previously, MV significantly
slows both MMP and MHC protein synthesis. Because MHC is the most predominant
protein in skeletal muscle it was hypothesized that 6 to 18 hours of MV would alter
pretranslational events in the diaphragm (i.e., MHC mRNA content would decrease). The
data, however, do not support this hypothesis.
As indicated in Figures 4-7 through 4-10, 6 to 18 hours of MV did not alter MHC
mRNA content in the diaphragm. These observations are consistent with previous studies
of locomotor skeletal muscle disuse. In the rat soleus 3-MHC (slow) mRNA content does
not significantly change after 5 hours or 7 days of hindlimb unloading, yet the synthetic
rate of MHC protein decreases significantly (162). Further, 6 to 72 hours of hindlimb
immobilization does not change the amount of a-actin mRNA in the rat gastrocnemius
but the synthetic rate of a-actin protein was decreased -65% during the first 6 hours
(173). The data from the current study in conjunction with previous studies demonstrates
that the rapid decrease in the rate of protein synthesis in the diaphragm, like other skeletal
muscles, is not due to a change in MHC mRNA content. Nonetheless, a recent report
indicates that extended periods of MV, >48 hours, does alter MHC mRNA (180). Using
Northern blot analysis, this group reported that >48 hours of MV increases MHC IIa
(70%) and MHC IIx (22%) mRNA, with little change in lib MHC mRNA (4%), and no
change in type I mRNA (180). Immunohistochemistry was used to fiber type a portion of
diaphragm after >48 hours of MV and detected a significant decrease in type I fibers and
a significant increase in fibers co-expressing both type I and II MHC protein (180). These
findings (180), in conjunction with the present study, suggest that during the first 18
hours of MV there is no measurable change in MHC mRNA but over the course of the
next 36 hours MHC mRNA expression does change and leads to a slow-to-fast MHC
shift associated with skeletal muscle unloading.
Regulation of Protein Synthesis
Protein synthesis is the culmination of many events, including transcription and
translation; all of which are highly regulated. The rapid decrease in protein synthesis after
MV could be due to the inhibition of one or both of these steps. A discussion of key
points of regulation of protein synthesis as they pertain to the MV-induced decrease in
protein synthesis follows.
In healthy active skeletal muscle, MHC protein expression appears to be regulated
by transcriptional events (18). For example, 3-MHC promoter region activity in the
soleus is significantly decreased after 7 days of inactivity (58, 80). Additionally, changes
in mRNA expression precede changes in protein expression measured from the 4th day to
the 90th day of inactivity (79). Thus, over a period of days/weeks/months MHC protein
expression is regulated by transcriptional events but during the first hours of reduced use
(e.g., 18 hours) protein expression is regulated by post-transcriptional events.
MV did not change the amount of total RNA (Table 4-5) or MHC mRNA (Figures
4-7 through 4-10). Hence, the observed decrease in the rate of protein synthesis without a
decrease in total RNA and MHC mRNA is indicative of a decrease in translational
efficiency (the amount of MHC protein synthesized per amount of MHC mRNA). A
decrease in translational efficiency occurs when one or more steps of translation is
The process of translating mRNA into a nascent polypeptide chain includes
initiation, elongation, and termination. In the following sections control of initiation will
be discussed in terms of relevant protein (initiation) factors and the pathway that controls
the assembly of the initiation complex. This will be followed by a discussion focusing on
the regulation of elongation and termination via the 3' end of mRNA.
Translation initiation is the result of a series of steps culminating in the 40S and
60S ribosomal subunits binding to mRNA. The regulatory processes involved in initiation
have been well elucidated. Specifically, proteins known as eukaryotic initiation factors
(elFs) are required for the engagement of the 40S ribosomal subunit and the 60S
ribosomal subunit with mRNA. elF function/activity is regulated by specific kinases and
phosphatases. Of particular interest is the regulation of eIF4E by 4E-binding protein
(BP)1. eIF4e is a critical component of the initiation complex and when bound by 4E-
BP1 initiation is hindered. 4E-BP1 binding of eIF4E is regulated by Akt (also known as
protein kinase B) and the putative kinase, mammalian target of rapammycin (mTOR)
(144). Akt and mTOR are central components of the Akt/mTOR pathway, which is
modulated by the loading state of skeletal muscle.
Akt acts directly on mTOR and the activity of Akt is sensitive to disuse. After 2
weeks of hindlimb suspension Akt protein expression and phosphorylated Akt (active
Akt) is significantly decreased (25). mTOR phosphorylation is modulated by Akt and 2
weeks of hindlimb suspension leads to a 60% decrease in mTOR phosphorylation (131).
Further, 2 weeks of hindlimb unloading increases 4E-BP 1 binding to eIF4E by >100%
(25). The results ofBodine et al. (25) and Reynolds et al. (131) suggest that hindlimb
unloading decreases the amount of Akt which in turn decreases mTOR activity leading to
increased 4E-BP1 binding to eIF4E (25) and thus preventing eIF4E from participating in
initiation. This is significant because it directly implicates the Akt/mTOR pathway in
muscle atrophy by inhibiting initiation.
In addition to controlling the phosphorylation state of 4E-BP 1, the Akt/mTOR
pathway controls the activity of the 70-kDa 40S ribosomal protein S6 kinase (p70S6k),
possibly through mTOR and directly by protein dependent kinase 1 (PDK1) (144). After
the phosphorylation of p70S6k its activity increases. Control of p70S6k is important
because this kinase controls the function of the ribosomal protein S6, a component of the
40S ribosomal subunit that is involved in tRNA recognition (144). After 12 hours of
hindlimb unloading or after 12 hours of denervation the phosphorylation state of p70S6k is
decreased 3-fold and remains depressed after 7 days (76). The decreased
phosphorylation state of p70s6k has also been reported after 2 weeks of hindlimb
suspension (25). Future experiments should explore the effect of MV on inhibition of
translation initiation in the diaphragm.
Translation Elongation and Termination
In addition to impairing initiation, reduced use of skeletal muscle impairs
elongation and termination. Previous studies (12, 88) indicate that during the initial hours
of reduced use protein synthesis in skeletal muscle stalls during elongation. Polysome
density is a measure of the number of ribosomes engaged in elongation per mRNA;
increasing in the number of ribosomes per mRNA increases the density of polysomes. Ku
and Thomason (88) studied a-actin polysome density after 18 hours of hindlimb
unloading and found a significant increase in polysome density. These data indicate that
assembly of the ribosomes on mRNA, (i.e., initiation) continues during the first 18 hours
of unloading. Further, increased polysome density indicates that elongation is somehow
As a follow up to the study of Ku and Thomason (88), Ashley and Russell (12)
tested the hypothesis that the 3' UTR of the j3-MHC regulates the decrease in protein
synthesis after 2 days of tenotomy in the rat soleus. Translation in the -3-MHC 3' UTR
was significantly decreased (12). Importantly, they found a significant increase in the
binding of a trans-acting protein factor in the 3' UTR (12). Based on these findings and
the work of Ku and Thomason (88) the authors suggest the following model: During
unloading the trans-acting protein binds to the 3-MHC mRNA 3' UTR with greater
affinity (12). This binding would physically prevent the ribosomes from reaching the stop
codon during elongation (i.e., translation would stall) (12). This would prevent a
completed protein from being released (termination) and thus repress protein expression
In summary, the MV-induced decrease in MMP and MHC protein synthesis in the
diaphragm may be due to alterations in the translational apparatus. Indeed, our finding
that MV does not alter diaphragmatic MHC mRNA levels but results in a decreased rate
of protein synthesis is consistent with this postulate. The current study did not measure
rates of initiation or elongation, inhibition of the Akt/mTOR pathway and/or ribosomal
stalling during elongation. However, one or more of these mechanisms may contribute to
the rapid decrease in protein synthesis induced by MV. This is an interesting area for
Critique of the Experimental Model
These experiments measured the changes in protein synthesis in the diaphragm
during the initial hours of MV. Due to the invasive nature of these experiments the rat
was used as the experimental model because of the biochemical and functional
similarities between the rat and human diaphragm. The rate of incorporation of the stable
tracer [13C]leucine into diaphragmatic proteins was used to measure protein synthesis. To
account for possible limitations of the experimental model a SB group was incorporated
into the experimental design. The SB animals underwent a surgical procedure identical to
the MV animals and received the same anesthetic for the same period of time. The SB
animals, therefore, served as time matched controls for the MV group so that any
alterations observed could be attributed to the effect MV. Of particular interest to these
experiments are the following considerations: the animals were not fed, the possibility
that anesthesia impacted protein synthesis, the length of the infusion period, and the
removal of blood.
The use of 13C[leucine] precluded feeding the MV and SB animals during the
experiments. 13C is a naturally occurring stable isotope present in all foods. Likewise,
leucine is a branched chain amino acid present in protein sources. Thus, feeding the
animals during the experiments would introduce an unknown amount of leucine with an
unknown amount of 13C and 12C into the animal's circulation and tissue (Kevin
Yarasheski, personal communication). This would dilute the 13C[leucine] tracer
administered to measure muscle protein synthesis by an unknown amount. Thus, the
interpretation of the 13C[leucine] enrichment data would be difficult if not impossible.
Therefore, to avoid confounding the 13C[leucine] enrichment measures, we chose not to
feed the MV and SB animals during the experimental period.
It was observed that protein synthesis decreased over time in the SB group.
Comparing the SB6 group to the SB 18 group the animals experienced a 32% decrease in
the rate of MMP synthesis and a 41% decrease in the rate of MHC synthesis. The
observed changes in the synthetic rate are consistent with the literature. Goldspink et al.
(62) report a 48% decrease in diaphragm MMP synthesis 23 hours post feeding and Bates
et al. (22) report a 44% decrease in the rate of limb-locomotor MHC synthesis 24 hours
post feeding. However, comparing the MV18 results to the time matched SB group a
29% decrease in MMP synthesis and a 68% decrease in MHC synthesis is still realized.
Therefore, the impact of MV on protein synthesis in the diaphragm was not obscured by
the nutrient status of the animals.
The anesthetic agent, sodium pentobarbital, could have impacted protein synthesis
in the diaphragm. Nonetheless, rats anesthetized with 20 mg/kg sodium pentobarbital
(twice the dose used in the current experiments) for 1 hour did not experience a
significant decrease in protein synthesis in skeletal muscle (74). Additionally, general
anesthesia does not decrease protein synthesis in skeletal muscle in healthy humans
undergoing abdominal surgery (47). Collectively, these experiments indicate that protein
synthesis is not altered by anesthesia per se. The influence of continued exposure of any
given anesthetic agent (e.g., 18 hours) would be difficult to separate from the reduced use
during that state. However, the experiments reviewed above (47, 74) report normal rates
of protein synthesis in limb-locomotor skeletal muscle during periods of time that
reduced use would not be expected to have an affect on protein synthesis. These reports
(47, 74) indicate that anesthesia does not affect protein synthesis; therefore, the decreased
rate of protein synthesis in the diaphragm during MV is attributable to MV, not the
Protein synthesis in the diaphragm was measured using the primed dose constant
infusion method with the stable tracer [13C]leucine. A plateau in 13C enrichment of the
plasma was achieved over the 6-hour infusion period (Figure 4-1). It is unknown if such a
plateau occurred within the tissue fluid of the diaphragm. The infusion period was 6
hours for all three experimental time points and tissue samples were taken at the
completion of the experimental period. Early work by Fern and Garlick (50)
demonstrated that a plateau in the enrichment of the plasma pool occurs within 2 hours
but enrichment of the labeled free amino acid in the tissue fluid of the diaphragm
continued to increase during 6 hours of infusion. The same authors (50) point out that if
the free amino acid pool in the plasma or tissue reflects the labeled protein precursor then
the calculated protein synthesis rate should be the same regardless of which precursor
pool is used in the calculation. Despite the continued rise in tissue fluid enrichment, the
authors (50) report similar rates of protein synthesis using each of the precursor pools,
with the plasma precursor pool giving the slowest rates. Similar to Fern and Garlick (50),
the rates of protein synthesis calculated using plasma [13C]leucine in the present
experiments underestimated the rates of protein synthesis. The concentration or
enrichment of [13C]leucine in the plasma should be greater than the enrichment of
"downstream" precursor pools such as tissue fluid and the transamination product of
leucine, KIC. Therefore, using plasma [13C]leucine as a precursor pool to calculate
protein synthesis should, and does, give a lower estimation of protein synthesis than
tissue fluid or KIC enrichment. The data in the present experiments indicate that plasma
[13C]KIC and tissue fluid [13C]leucine are appropriate surrogates of t-leucyl RNA.
Indeed, using these surrogates to calculate rates of protein synthesis yields similar rates
The duration of the [13C]leucine infusion period was 6 hours. This duration was
chosen in order to achieve a plateau in [13C]leucine enrichment of the plasma precursor
pool (see Figure 4-1). Due to the length of the infusion period the rates of protein
synthesis are a rolling average of the 6-hour period. Thus, the rates of protein synthesis at
each time point may underestimate the actual rate of protein synthesis at any given
moment. This would be the most profound during the first 6 hours of MV, as the last hour
would be averaged in with the first.
One milliliter of blood was drawn from each animal before [13C]leucine infusion
and after the 5th and 6th hours of infusion. Following each blood draw an equal volume of
normal saline was administered to prevent hypovolemia. The initial blood sample and the
sample taken after the 5th hour precluded our ability to monitor arterial blood gas during
the experiments. Nonetheless, we have demonstrated that our MV protocol results in only
minor disturbances in blood gas homeostasis over a 24-hour period (125). However, SB
animals typically experience some degree of respiratory acidosis without any significant
affect on diaphragmatic contractile function (125). During the present experiments we
relied on our previous experience with the mechanical ventilator and anesthesia
parameters and it is possible that blood gas homeostasis was not adequately maintained.
It should be noted that over the course of these experiments a high degree of surgical
success was achieved (i.e., 88% of the experiments were successful) suggesting that
animal homeostasis was well maintained despite our inability to monitor blood gas
Mode of Mechanical Ventilation
Pressure-assist MV is commonly used to treat adult patients in intensive care units.
However, we used controlled MV for two reasons. First, because controlled MV results
in rapid diaphragmatic atrophy (167) the impact of controlled MV can be studied during
relatively short time periods. Second, controlled MV is clinically relevant as it is used in
adult patients following drug over dose, spinal cord injury and is commonly used in
certain pediatric situations (72).
Summary and Future Experiments
These experiments investigated the affect of MV on protein synthesis and MHC
mRNA in the rat diaphragm. The hypothesis that MV-induced diaphragmatic atrophy is,
at least in part, due to a decreased rate of total (MMP) and myofibrillar (MHC) protein
synthesis was supported. However, the hypothesis that MV alters pretranslational events
in the diaphragm was not supported.
Future experiments investigating the mechanisms that regulate protein synthesis in
the diaphragm during MV should follow several pathways. First, the role the Akt/mTOR
pathway plays in regulating the synthesis of diaphragmatic proteins during MV should be
studied. Secondly, polysome density of actin and myosin mRNAs after MV should be
measured to determine if these mRNAs are acutely regulated by arresting translation
during elongation. Additionally, identification of the trans-acting protein(s) binding to
the MHC mRNA 3' UTR should be pursued.
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